Nectar-secreting floral stomata in Maxillaria anceps Ames & C. Schweinf. (Orchidaceae).

PubMed

Davies, K L; Stpiczyńska, M; Gregg, A

2005-08-01

Although it was generally assumed that Maxillaria spp. do not produce nectar, in recent years, nectar has been reported for a number of these orchids. Nevertheless, our current understanding of nectary structure and nectar secretion in Maxillaria is based solely on M. coccinea (Jacq.) L.O. Williams ex Hodge, which, since it shows many features characteristic of ornithophilous flowers, is atypical of this largely entomophilous genus. The aim of the present paper is to describe, for the first time, nectar secretion in a presumed entomophilous species of Maxillaria. The structure of the nectary of M. anceps Ames & C. Schweinf., nectar composition and the process of nectar secretion were investigated using light microscopy, scanning electron microscopy, transmission electron microscopy, histochemistry, refractometry and high performance liquid chromatography. Nectar appears as droplets that are exuded by modified stomata borne upon the labellar callus and collects upon the labellum and at the base of the column-foot. Although such stomata are known to occur in a number of angiosperm families, this is the first time for them to be observed in orchids. The callus consists largely of parenchyma with raphides and is supplied by eight to ten collateral bundles. This tissue, together with the single-layered epidermis, seemingly contains terpenoids. During the bud stage, the callus cells contain an organelle complement consistent with secretory cells whereas by day 4 of anthesis, much of the cell is occupied by a vacuole. The nectar is sucrose-dominant but also contains low concentrations of glucose, fructose, free amino acids and possibly terpenoids. The high sugar concentration (approx. 66 %) is consistent with melittophily and may indicate that, like the majority of Maxillaria spp., M. anceps is visited by stingless bees (Meliponini).

* Composite Biomaterial as a Carrier for Bone-Active Substances for Metaphyseal Tibial Bone Defect Reconstruction in Rats.

PubMed

Horstmann, Peter Frederik; Raina, Deepak Bushan; Isaksson, Hanna; Hettwer, Werner; Lidgren, Lars; Petersen, Michael Mørk; Tägil, Magnus

2017-12-01

Restoring lost bone is a major challenge in orthopedic surgery. Currently available treatment strategies have shortcomings, such as risk of infection, nonunion, and excessive resorption. Our primary aim was to study if a commercially available gentamicin-containing composite calcium sulfate/hydroxyapatite biomaterial (GBM) could serve as a carrier for local delivery of bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA) in a tibia defect model in rats. Empty and allograft-filled defects were used as controls. A 3 × 4-mm metaphyseal bone defect was created in the proximal tibia, and the rats were grouped according to defect filling: (1) Empty, (2) Allograft, (3) GBM, (4) GBM + ZA, and (5) GBM + ZA + BMP-2. In vivo microcomputed tomography (micro-CT) images at 4 weeks showed significantly higher mineralized tissue volume (MV) in the intramedullary defect region and the neocortical/callus region in all GBM-treated groups. After euthanization at 8 weeks, ex vivo micro-CT showed that addition of ZA (GBM + ZA) and BMP-2 (GBM + ZA + BMP-2) mainly increased the neocortical and callus formation, with the highest MV in the combined ZA and BMP-2-treated group. Qualitative histological analysis, verifying the increased neocortical/callus thickness and finding of trabecular bone in all GBM-treated groups, supported that the differences in MV measured with micro-CT in fact represented bone tissue. In conclusion, GBM can serve as a carrier for ZA and BMP-2 leading to increased MV in the neocortex and callus of a metaphyseal bone defect in rats.

[Preliminary result of allogenic bone and autogeneic-iliac bone in comminuted fracture reparation in rabbits].

PubMed

Wang, Zhi-qiang; Li, Qi-jia; Wang, Qi

2002-11-01

To observe the difference of the fracture reparation using autogeneic-iliac bone and allogenic bone. Comminuted fracture of humerus in two sides were made in rabbits. Autogeneic-iliac bone was implanted in one side, while allogenic bone of equal capacity was implanted in the other side. General observation, X-ray, and HE histologic section were done when the rabbits were put to death in different stages. One week after implantation, the graft had been enclosed by connective tissue without infiltration of the inflammatory cells. At the 2nd week, the graft had been enclosed in osteoplastic granulation tissue, and the cartilage callus had formed. At the 3rd week, there had been broken sequestrum among the callus; the cartilage had actively formed the bone; and the medulla had been making. At the 4th week, the sequestrum had disappeared, and the mature callus had appeared; the osteoblasts had arranged in a line around the edge of the mature callus. At the 5th week, the callus was strong, compact and approached mature bones. At the 6th week, there had been the compact lamellar structures and the complete haversian's systems. There was no significant difference between callus of two sides by using image quantitative analysis in the 3rd, 4th week (P > 0.05). The allogenic bone has good histocompatibility and bone conduction effect, and can be used for bone transplantation substitute with autogenous-iliac bone.

Somatic embryogenesis from flower explants of cocoa (Theobroma cacao L.).

PubMed

Silva, J J; Debergh, P

2001-01-01

Two types of flower explants, staminoides and petals, were used for in vitro induction of somatic embryos in cocoa. After 14 days in culture, we observed globular structures and callus formation on both types of explants. However, the better results were obtained on staminoides: 98.3% formed callus and 86.2% somatic embryos on Murashige and Skoog (1962) medium supplemented with sucrose, coconut water, 2,4-D, kinetin and agar.

The effect of light quality on the pro-/antioxidant balance, activity of photosystem II, and expression of light-dependent genes in Eutrema salsugineum callus cells.

PubMed

Pashkovskiy, P P; Soshinkova, T N; Korolkova, D V; Kartashov, A V; Zlobin, I E; Lyubimov, V Yu; Kreslavski, V D; Kuznetsov, Vl V

2018-05-01

The antioxidant balance, photochemical activity of photosystem II (PSII), and photosynthetic pigment content, as well as the expression of genes involved in the light signalling of callus lines of Eutrema salsugineum plants (earlier Thellungiella salsuginea) under different spectral light compositions were studied. Growth of callus in red light (RL, maximum 660 nm), in contrast to blue light (BL, maximum 450 nm), resulted in a lower H 2 O 2 content and thiobarbituric acid reactive substances (TBARS). The BL increased the activities of key antioxidant enzymes in comparison with the white light (WL) and RL and demonstrated the minimum level of PSII photochemical activity. The activities of catalase (CAT) and peroxidase (POD) had the highest values in BL, which, along with the increased H 2 O 2 and TBARS content, indicate a higher level of oxidative stress in the cells. The expression levels of the main chloroplast protein genes of PSII (PSBA and PSBD), the NADPH-dependent oxidase gene of the plasma membrane (RbohD), the protochlorophyllide oxidoreductase genes (POR B, C) involved in the biosynthesis of chlorophyll, and the key photoreceptor signalling genes (CIB1, CRY2, PhyB, PhyA, and PIF3) were determined. Possible mechanisms of light quality effects on the physiological parameters of callus cells are discussed.

Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair

PubMed Central

Reumann, Marie K.; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Steven B.; Lukashova, Lyudmila; Boskey, Adele L.; Mayer-Kuckuk, Philipp

2011-01-01

Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1−/− mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1−/− mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1−/− callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair. PMID:21726677

Loss of transcription factor early growth response gene 1 results in impaired endochondral bone repair.

PubMed

Reumann, Marie K; Strachna, Olga; Yagerman, Sarah; Torrecilla, Daniel; Kim, Jihye; Doty, Stephen B; Lukashova, Lyudmila; Boskey, Adele L; Mayer-Kuckuk, Philipp

2011-10-01

Transcription factors that play a role in ossification during development are expected to participate in postnatal fracture repair since the endochondral bone formation that occurs in embryos is recapitulated during fracture repair. However, inherent differences exist between bone development and fracture repair, including a sudden disruption of tissue integrity followed by an inflammatory response. This raises the possibility that repair-specific transcription factors participate in bone healing. Here, we assessed the consequence of loss of early growth response gene 1 (EGR-1) on endochondral bone healing because this transcription factor has been shown to modulate repair in vascularized tissues. Model fractures were created in ribs of wild type (wt) and EGR-1(-/-) mice. Differences in tissue morphology and composition between these two animal groups were followed over 28 post fracture days (PFDs). In wt mice, bone healing occurred in healing phases characteristic of endochondral bone repair. A similar healing sequence was observed in EGR-1(-/-) mice but was impaired by alterations. A persistent accumulation of fibrin between the disconnected bones was observed on PFD7 and remained pronounced in the callus on PFD14. Additionally, the PFD14 callus was abnormally enlarged and showed increased deposition of mineralized tissue. Cartilage ossification in the callus was associated with hyper-vascularity and -proliferation. Moreover, cell deposits located in proximity to the callus within skeletal muscle were detected on PFD14. Despite these impairments, repair in EGR-1(-/-) callus advanced on PFD28, suggesting EGR-1 is not essential for healing. Together, this study provides genetic evidence that EGR-1 is a pleiotropic regulator of endochondral fracture repair. Copyright © 2011 Elsevier Inc. All rights reserved.

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content.

PubMed

Fortes, A M; Pais, M S

2000-07-01

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.

Blisters, Calluses, and Corns

MedlinePlus

... for Educators Search English Español Blisters, Calluses, and Corns KidsHealth / For Kids / Blisters, Calluses, and Corns What's ... used to all of that stress. What's a Corn? Like calluses, corns are also areas of hard, ...

Metabolomic homeostasis shifts after callus formation and shoot regeneration in tomato

PubMed Central

Kumari, Alka; Ray, Kamalika; Sadhna, Sadhna; Pandey, Arun Kumar; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

2017-01-01

Plants can regenerate from a variety of tissues on culturing in appropriate media. However, the metabolic shifts involved in callus formation and shoot regeneration are largely unknown. The metabolic profiles of callus generated from tomato (Solanum lycopersicum) cotyledons and that of shoot regenerated from callus were compared with the pct1-2 mutant that exhibits enhanced polar auxin transport and the shr mutant that exhibits elevated nitric oxide levels. The transformation from cotyledon to callus involved a major shift in metabolite profiles with denser metabolic networks in the callus. In contrast, the transformation from callus to shoot involved minor changes in the networks. The metabolic networks in pct1-2 and shr mutants were distinct from wild type and were rewired with shifts in endogenous hormones and metabolite interactions. The callus formation was accompanied by a reduction in the levels of metabolites involved in cell wall lignification and cellular immunity. On the contrary, the levels of monoamines were upregulated in the callus and regenerated shoot. The callus formation and shoot regeneration were accompanied by an increase in salicylic acid in wild type and mutants. The transformation to the callus and also to the shoot downregulated LST8 and upregulated TOR transcript levels indicating a putative linkage between metabolic shift and TOR signalling pathway. The network analysis indicates that shift in metabolite profiles during callus formation and shoot regeneration is governed by a complex interaction between metabolites and endogenous hormones. PMID:28481937

Callus features of regenerate fracture cases in femoral lengthening in achondroplasia.

PubMed

Devmurari, Kamlesh N; Song, Hae Ryong; Modi, Hitesh N; Venkatesh, K P; Ju, Kim Seung; Song, Sang Heon

2010-09-01

We studied the callus features seen in cases of regenerate fracture in femoral lengthening using a monolateral fixator in achondroplasia to determine whether callus types and shapes can predict the probability of callus fracture. The radiographs of 28 cases of femoral lengthening in 14 patients, 14 cases of callus fracture, and 14 cases without callus fracture were retrospectively analyzed by four observers and classified into different shapes and types in concordance with the Ru Li classification. The average lengthening of 9.4 cm (range 7.5-11.8 cm) was achieved, which was 41% (range 30-55%) of the original length and the average timing of callus fracture was 470 days (range 440-545 days) after surgery in the callus fracture group. While the average lengthening of 9.1 cm (range 8-9.7 cm) was achieved, this was 30% (range 28-32%) of the original length in the group of patients without callus fracture. The callus was atypically shaped, there was a 48% average (range 30-72%) reduction of the callus width compared with the natural width of the femur, and a lucent pathway was present in all cases of regenerate fracture. A lucent pathway was seen in all fracture cases with concave, lateral, and atypical shapes, and there was more than 30% lengthening and 30% reduction of the callus width compared with the natural width of the femur, which are the warning signs for regenerate fractures. These signs help the surgeon to predict the outcome and guide him in planning for any additional interventions. The Ru Li classification is an effective method for the evaluation of the chance of callus fracture.

Callus induction and flavonoid production on the immature seed of Stelechocarpus burahol

NASA Astrophysics Data System (ADS)

Habibah, N. A.; Moeljopawiro, S.; Dewi, K.; Indrianto, A.

2018-03-01

Stelechocarpus burahol [(Bl.) Hook. f. & Th.] is one of the medicinal plants. In vitro callus induction studies on S. burahol were carried out to determine phytohormone requirement for optimum callus induction. Immature seed explants were cultured on MS medium by adding different kinds and different concentrations of plant growth regulators (picloram and 2,4-D) under light and dark conditions. The results showed that callus formation was initiated on the 18,50th to the 55th days. The best condition for optimum callus induction was found on MS medium, which was supplemented with 7.5 mg/L picloram and was maintained in the dark condition. The callus induction varied from 60% to 100%. The callus that produced the highest flavonoid was grown on the medium with the addition of 10 mg/L of 2,4-D. In conclusion, the results represented a suitable medium for S.burahol callus induction.

Local administration of a hedgehog agonist accelerates fracture healing in a mouse model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kashiwagi, Miki; Division of Clinical Biotechnology, The University of Tokyo Graduate School of Medicine, Bunkyo-ku, Tokyo, 113-0033; Hojo, Hironori

Bone fracture healing is processed through multiple biological stages including the transition from cartilaginous callus to bony callus formation. Because of its specific, temporal and indispensable functions demonstrated by mouse genetic studies, Hedgehog (Hh) signaling is one of the most potent signaling pathways involved in these processes, but the effect of Hh-signaling activation by small compounds on the repair process had not yet been addressed. Here we examined therapeutic effects of local and one shot-administration of the Hh agonist known as smoothened agonist (SAG) on bone fracture healing in a mouse model. A quantitative analysis with three-dimensional micro-computed tomography showedmore » that SAG administration increased the size of both the cartilaginous callus and bony callus at 14 days after the surgery. A histological analysis showed that SAG administration increased the number of cells expressing a proliferation marker and a chondrocyte marker in cartilaginous callus as well as the cells expressing an osteoblast marker in bony callus. These results indicate that the SAG administration resulted in an enhancement of callus formation during bone fracture healing, which is at least in part mediated by an increase in chondrocyte proliferation in cartilaginous callus and the promotion of bone formation in bony callus. Therapeutic strategies with a SAG-mediated protocol may thus be useful for the treatment of bone fractures. - Highlights: • Local administration of a Hh agonist accelerates callus formation. • The Hh agonist administration promotes chondrocyte proliferation in the soft callus. • The Hh agonist administration increases osteoblast formation in the hard callus.« less

Factors Associated With Callus in Patients with Diabetes, Focused on Plantar Shear Stress During Gait.

PubMed

Hamatani, Masako; Mori, Taketoshi; Oe, Makoto; Noguchi, Hiroshi; Takehara, Kimie; Amemiya, Ayumi; Ohashi, Yumiko; Ueki, Kohjiro; Kadowaki, Takashi; Sanada, Hiromi

2016-11-01

The aim of this study is to identify whether plantar shear stress in neuropathic patients with diabetes with callus is increased compared with those without callus. The differences in foot deformity, limited joint mobility, repetitive stress of walking, and ill-fitting shoes between patients with callus and those without callus were also determined. Subjects were recruited from the Diabetic Foot Outpatient Clinic. A newly developed in-shoe measurement system, which has flexible and thin insoles, enabled measurement of both plantar pressure and shear stress simultaneously when subjects walked as usual on a 10 m walkway. It was found that plantar shear stress adjusted for weight during the push-off phase was increased by 1.32 times in patients with callus compared with those without callus (mean ± SD: 0.0500 ± 0.0160 vs 0.0380 ± 0.0144, P = .031). Moreover, hallux valgus deformity, reduction in dorsiflexion of the ankle joint and increase in plantar flexion were showed in feet with callus. Increased plantar shear stress may be caused by gait change that patients having callus push off with the metatarsal head instead of the toe as a result of foot deformity and limited joint mobility. It was found that plantar shear stress adjusted for weight during the push-off phase was increased in patients with callus compared with those without callus by using the newly developed measurement system. These results suggest that reduction of plantar shear stress during the push-off phase can prevent callus formation in neuropathic patients with diabetes. © 2016 Diabetes Technology Society.

NMR-based metabolomics study of the biochemical relationship between sugarcane callus tissues and their respective nutrient culture media

PubMed Central

Mahmud, Iqbal; Thapaliya, Monica; Boroujerdi, Arezue; Chowdhury, Kamal

2014-01-01

The culture of sugarcane leaf explant onto culture induction medium triggers the stimulation of cell metabolism into both embryogenic and non-embryogenic callus tissues. Previous analyses demonstrated that embryogenic and nonembryogenic callus tissues have distinct metabolic profiles. This study is the follow-up to understand the biochemical relationship between the nutrient media and callus tissues using one-dimensional (1D 1H) and two-dimensional (2D 1H–13C) NMR spectroscopy followed by principal component analysis (PCA). 1D 1H spectral comparisons of fresh unspent media (FM), embryogenic callus media (ECM), non-embryogenic callus media (NECM), embryogenic callus (EC), and non-embryogenic callus (NEC), showed different metabolic relationships between callus tissues and media. Based on metabolite fold change analysis, significantly changing sugar compounds such as glucose, fructose, sucrose, and maltose were maintained in large quantities by EC only. Significantly different amino acid compounds such as valine, leucine, alanine, threonine, asparagine, and glutamine and different organic acid derivatives such as lactate, 2-hydroxyisobutyrate, 4-aminobutyrate, malonate, and choline were present in EC, NEC, and NECM, which indicates that EC maintained these nutrients, while NEC either maintained or secreted the metabolites. These media and callus-specific results suggest that EC and NEC utilize and/or secrete media nutrients differently. PMID:25012359

A comparison of stereology, structural rigidity and a novel 3D failure surface analysis method in the assessment of torsional strength and stiffness in a mouse tibia fracture model.

PubMed

Wright, David A; Nam, Diane; Whyne, Cari M

2012-08-31

In attempting to develop non-invasive image based measures for the determination of the biomechanical integrity of healing fractures, traditional μCT based measurements have been limited. This study presents the development and evaluation of a tool for assessment of fracture callus mechanical properties through determination of the geometric characteristics of the fracture callus, specifically along the surface of failure identified during destructive mechanical testing. Fractures were created in tibias of ten male mice and subjected to μCT imaging and biomechanical torsion testing. Failure surface analysis, along with previously described image based measures was calculated using the μCT image data, and correlated with mechanical strength and stiffness. Three-dimensional measures along the surface of failure, specifically the surface area and torsional rigidity of bone, were shown to be significantly correlating with mechanical strength and stiffness. It was also shown that surface area of bone along the failure surface exhibits stronger correlations with both strength and stiffness than measures of average and minimum torsional rigidity of the entire callus. Failure surfaces observed in this study were generally oriented at 45° to the long axis of the bone, and were not contained exclusively within the callus. This work represents a proof of concept study, and shows the potential utility of failure surface analysis in the assessment of fracture callus stability. Copyright © 2012 Elsevier Ltd. All rights reserved.

Somatic hybridization of sexually incompatible petunias: Petunia parodii, Petunia parviflora.

PubMed

Power, J B; Berry, S F; Chapman, J V; Cocking, E C

1980-01-01

Somatic hybrid plants were regenerated following the fusion of leaf mesophyll protoplasts of P. parodii with those isolated from a nuclear-albino mutant of P. parviflora. Attempts at sexual hybridization of these two species repeatedly failed thus confirming their previously established cross-incompatibility. Selection of somatic hybrid plants was possible since protoplasts of P. parodii would not develop beyond the cell colony stage, whilst those of the somatic hybrid and albino P. parviflora produced calluses. Green somatic hybrid calluses were visible against a background of albino cells/calluses, and upon transfer to regeneration media gave rise to shoots. Shoots and the resultant flowering plants were confirmed as somatic hybrids based on their growth habit, floral pigmentation and morphology, leaf hair structure, chromosome number and Fraction 1 protein profiles. The relevance of such hybrid material for the development of new, and extensively modified cultivars, is discussed.

Barley callus: a model system for bioengineering of starch in cereals.

PubMed

Carciofi, Massimiliano; Blennow, Andreas; Nielsen, Morten M; Holm, Preben B; Hebelstrup, Kim H

2012-09-07

Starch is the most important source of calories for human nutrition and the majority of it is produced by cereal farming. Starch is also used as a renewable raw material in a range of industrial sectors. It can be chemically modified to introduce new physicochemical properties. In this way starch is adapted to a variety of specific end-uses. Recombinant DNA technologies offers an alternative to starch industrial processing. The plant biosynthetic pathway can be manipulated to design starches with novel structure and improved technological properties. In the future this may reduce or eliminate the economical and environmental costs of industrial modification. Recently, many advances have been achieved to clarify the genetic mechanism that controls starch biosynthesis. Several genes involved in the synthesis and modification of complex carbohydrates in many organisms have been identified and cloned. This knowledge suggests a number of strategies and a series of candidate genes for genetic transformation of crops to generate new types of starch-based polymers. However transformation of cereals is a slow process and there is no easy model system available to test the efficiency of candidate genes in planta. We explored the possibility to use transgenic barley callus generated from immature embryo for a fast test of transgenic modification strategies of starch biosynthesis. We found that this callus contains 4% (w/w dw) starch granules, which we could modify by generating fully transgenic calli by Agrobacterium-transformation. A Green Fluorescent Protein reporter protein tag was used to identify and propagate only fully transgenic callus explants. Around 1 - 1.5 g dry weight of fully transgenic callus could be produced in 9 weeks. Callus starch granules were smaller than endosperm starch granules and contained less amylose. Similarly the expression profile of starch biosynthesis genes were slightly different in callus compared with developing endosperm. In this study we have developed an easy and rapid in planta model system for starch bioengineering in cereals. We suggest that this method can be used as a time-efficient model system for fast screening of candidate genes for the generation of modified starch or new types of carbohydrate polymers.

Barley callus: a model system for bioengineering of starch in cereals

PubMed Central

2012-01-01

Background Starch is the most important source of calories for human nutrition and the majority of it is produced by cereal farming. Starch is also used as a renewable raw material in a range of industrial sectors. It can be chemically modified to introduce new physicochemical properties. In this way starch is adapted to a variety of specific end-uses. Recombinant DNA technologies offers an alternative to starch industrial processing. The plant biosynthetic pathway can be manipulated to design starches with novel structure and improved technological properties. In the future this may reduce or eliminate the economical and environmental costs of industrial modification. Recently, many advances have been achieved to clarify the genetic mechanism that controls starch biosynthesis. Several genes involved in the synthesis and modification of complex carbohydrates in many organisms have been identified and cloned. This knowledge suggests a number of strategies and a series of candidate genes for genetic transformation of crops to generate new types of starch-based polymers. However transformation of cereals is a slow process and there is no easy model system available to test the efficiency of candidate genes in planta. Results We explored the possibility to use transgenic barley callus generated from immature embryo for a fast test of transgenic modification strategies of starch biosynthesis. We found that this callus contains 4% (w/w dw) starch granules, which we could modify by generating fully transgenic calli by Agrobacterium-transformation. A Green Fluorescent Protein reporter protein tag was used to identify and propagate only fully transgenic callus explants. Around 1 – 1.5 g dry weight of fully transgenic callus could be produced in 9 weeks. Callus starch granules were smaller than endosperm starch granules and contained less amylose. Similarly the expression profile of starch biosynthesis genes were slightly different in callus compared with developing endosperm. Conclusions In this study we have developed an easy and rapid in planta model system for starch bioengineering in cereals. We suggest that this method can be used as a time-efficient model system for fast screening of candidate genes for the generation of modified starch or new types of carbohydrate polymers. PMID:22958600

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl.

PubMed

Shriram, Varsha; Nanekar, Vikas; Kumar, Vinay; Kavi Kishor, P B

2014-11-01

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog's (MS) medium containing 2.5 mg L(-1) 6-benzylaminopurine (BAP), 1.0 mg L(-1) kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L(-1) each and 50 mg L(-1) ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and approximately 40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L(-1) CH, 2.5 mg L(-1) BAP and 1.0 mg L(-1) Kin with 30 PLBs and 6 shoots per callus mass (approximately 5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L(-1) indole-3-butyric acid, 200 mg L(-1) activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

Development of western spruce budworm on Douglas-fir callus tissue.

Treesearch

Roy C. Beckwith; Barry Goldfarb

1991-01-01

The success of feeding and development of western spruce budworm (Choristoneura occidentalis Freeman) on callus tissue of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) was determined. Fewer insects died when fed pure callus tissue than when fed on standard diet or callus incorporated into the standard diet. The final...

Ontogeny of plants under various gravity condition

NASA Astrophysics Data System (ADS)

Laurinavičius, R.; Švegždienṡ, D.; Raklevičienė, D.; Kenstavičienė, P.

2001-01-01

The results of experiments performed under conditions of microgravity (MG) or under its simulation on the horizontal clinostat (HC) with the callus, seedlings of various species and embryogenic structures have revealed a definite role of gravity as an ecological factor in the processes of cytomorphogenesis, growth, and development. The transformation of differentiated somatic cells of arabidopsis seed into undifferentiated callus was not inhibited under MG, though modifications of the whole callus morphology and of mean cell and nucleus size were observed. The morphogenesis of polar structures such as root-hair bearing cells of Lactuca primary root has been shown to be modified in the course of differentiation under mass acceleration diminished below 0.1 g. Seed germination and seedling morphogenesis under MG follow their normal course, but a significant stimulation of shoot growth with no effect on primary root growth has been determined. A successful in vitro regeneration of Nicotiana tabacum plantlets from leaf cells and subsequent formation of shoots and roots on a continuously rotating HC as well as the formation of viable seeds during seed-to-seed growth of Arabidopsis plants under MG have indicated that gravity plays but a limited role in the processes of embryogenesis and organogenesis.

Effect of an ozone injury-retardant chemical on isozyme profiles from alfalfa callus in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rier, J.P.; Sood, V.K.; Whitaker, A.

1983-01-01

Plant ozone injury retardant (EDU or ethylenediurea) at 1.0 ppm inhibited growth of callus of alfalfa cultivars Williamsburg (ozone-sensitive) and MSB-CW5An2(ozone-insensitive) germplasm of Medicago sative. The presence of EDU(0.1 ppm) in growth medium increased the number of protein and peroxidase isozyme bands in alfalfa cultivar stem callus and ozone modified their intensities. Protein profiles of MSB stem callus from media containing EDU or exposed to ozone were unchanged. Marked differences were observed between the peroxidase profiles of ozonated and control ozone-insensitive stem callus from media containing EDU. Protein profiles of ozonated ozone-insensitive leaf callus differed slightly from controls.

Morphohistobiochemical characteristics of embryogenic and nonembryogenic callus cultures of sweet potato (Ipomoea batatas L.).

PubMed

Mukherjee, A; Debata, B K; Mukherjee, P S; Malik, S K

2001-01-01

Ipomoea batatas callus culture raised in a medium supplemented with 2,4-D (2,4-dichlorophenoxy acetic acid) alone or 2,4-D in combination with benzyl adenine, were found to be embryogenic. Supplementation of exogenous chemicals, such as 5 g/l NaCI or 0.7 g/l proline together with a mild dose of 0.2 mg/l 2,4-D, enhanced somatic embryogenesis significantly in all the genotypes tested. Morphological, growth, physiological, histological, and biochemical characteristics of the embryogenic callus were different from the nonembryogenic callus. The former was compact, slow growing, and nodular compared with the fast growing, fragile, nonembryogenic callus. The embryogenic callus tissue had more dry matter, protein and reducing sugar contents compared with the less embryogenic callus. The somatic embryogenic response remained steady in the cultures for up to 96 weeks.

21 CFR 358.510 - Corn and callus remover active ingredients.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 5 2013-04-01 2013-04-01 false Corn and callus remover active ingredients. 358... USE Corn and Callus Remover Drug Products § 358.510 Corn and callus remover active ingredients. The product consists of any of the following active ingredients within the specified concentrations and in the...

21 CFR 358.510 - Corn and callus remover active ingredients.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 5 2014-04-01 2014-04-01 false Corn and callus remover active ingredients. 358... USE Corn and Callus Remover Drug Products § 358.510 Corn and callus remover active ingredients. The product consists of any of the following active ingredients within the specified concentrations and in the...

21 CFR 358.510 - Corn and callus remover active ingredients.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 5 2011-04-01 2011-04-01 false Corn and callus remover active ingredients. 358... USE Corn and Callus Remover Drug Products § 358.510 Corn and callus remover active ingredients. The product consists of any of the following active ingredients within the specified concentrations and in the...

21 CFR 358.510 - Corn and callus remover active ingredients.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 5 2012-04-01 2012-04-01 false Corn and callus remover active ingredients. 358... USE Corn and Callus Remover Drug Products § 358.510 Corn and callus remover active ingredients. The product consists of any of the following active ingredients within the specified concentrations and in the...

21 CFR 358.510 - Corn and callus remover active ingredients.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Corn and callus remover active ingredients. 358.510 Section 358.510 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... USE Corn and Callus Remover Drug Products § 358.510 Corn and callus remover active ingredients. The...

Alpha-glucosidase Inhibitory and Antioxidant Potential of Antidiabetic Herb Alternanthera sessilis: Comparative Analyses of Leaf and Callus Solvent Fractions.

PubMed

Chai, Tsun-Thai; Khoo, Chee-Siong; Tee, Chong-Siang; Wong, Fai-Chu

2016-01-01

Alternanthera sessilis is a medicinal herb which is consumed as vegetable and used as traditional remedies of various ailments in Asia and Africa. This study aimed to investigate the antiglucosidase and antioxidant activity of solvent fractions of A. sessilis leaf and callus. Leaf and callus methanol extracts were fractionated to produce hexane, chloroform, ethyl acetate, butanol, and water fractions. Antiglucosidase and 1,1-diphenyl-2-picrylhydrazyl scavenging activities as well as total phenolic (TP), total flavonoid (TF), and total coumarin (TC) contents were evaluated. Lineweaver-Burk plot analysis was performed on leaf and callus fractions with the strongest antiglucosidase activity. Leaf ethyl acetate fraction (LEF) had the strongest antiglucosidase (EC 50 0.55 mg/mL) and radical scavenging (EC 50 10.81 μg/mL) activity among leaf fractions. Callus ethyl acetate fraction (CEF) and chloroform fraction had the highest antiglucosidase (EC 50 0.25 mg/mL) and radical scavenging (EC 50 34.12 μg/mL) activity, respectively, among callus fractions. LEF and CEF were identified as noncompetitive and competitive α-glucosidase inhibitors, respectively. LEF and CEF had greater antiglucosidase activity than acarbose. Leaf fractions had higher phytochemical contents than callus fractions. LEF had the highest TP, TF, and TC contents. Antiglucosidase and antioxidant activities of leaf fractions correlated with phytochemical contents. LEF had potent antiglucosidase activity and concurrent antioxidant activity. CEF had the highest antiglucosidase activity among all fractions. Callus culture is a promising tool for enhancing production of potent α-glucosidase inhibitors. Leaf ethyl acetate fraction (LEF) had the strongest antiglucosidase (EC 50 0.55 mg/mL) and radical scavenging (EC 50 10.81 μg/mL) activity among leaf fractionsCallus ethyl acetate fraction (CEF) and chloroform fraction had the highest antiglucosidase (EC 50 0.25 mg/mL) and radical scavenging (EC 50 34.12 μg/mL) activity, respectively, among callus fractionsLEF and CEF were identified as noncompetitive and competitive á-glucosidase inhibitors, respectivelyAntiglucosidase and antioxidant activities of leaf fractions correlated with phytochemical contents. Abbreviations used: LHF: Leaf hexane fraction, LCF: Leaf chloroform fraction, LEF: Leaf ethyl acetate fraction, LBF: Leaf butanol fraction, LWF: Leaf water fraction, CHF: Callus hexane fraction, CCF: Callus chloroform fraction, CEF: Callus ethyl acetate fraction, CBF: Callus butanol fraction, CWF: Callus water fraction, TP: Total phenolic, TF: Total flavonoid, TC: Total coumarin.

Callus remodelling model

NASA Astrophysics Data System (ADS)

Miodowska, Justyna; Bielski, Jan; Kromka-Szydek, Magdalena

2018-01-01

The objective of this paper is to investigate the healing process of the callus using bone remodelling approach. A new mathematical model of bone remodelling is proposed including both underload and overload resorption, as well as equilibrium and bone growth states. The created model is used to predict the stress-stimulated change in the callus density. The permanent and intermittent loading programs are considered. The analyses indicate that obtaining a sufficiently high values of the callus density (and hence the elasticity) modulus is only possible using time-varying load parameters. The model predictions also show that intermittent loading program causes delayed callus healing. Understanding how mechanical conditions influence callus remodelling process may be relevant in the bone fracture treatment and initial bone loading during rehabilitation.

[Chromosome variability in the tissue culture of rare Gentiana species].

PubMed

Tvardovs'ka, M O; Strashniuk, N M; Mel'nyk, V M; Adonin, V I; Kunakh, V A

2008-01-01

Cytogenetic analysis of plants and tissue culture of Gentiana lutea, G. punctata, G. acaulis has been carried out. Culturing in vitro was found to result in the changes of chromosome number in the calluses of the species involved. Species specificity for variation of the cultured cell genomes was shown. Contribution of the original plant genotypes to the cytogenetic structure of the tissue culture was established. Gentiana callus tissues (except for in vitro culture of G. punctata, derived from plant of Breskul'ska population) were found to exhibit modal class with the cells of diploid and nearly diploid chromosome sets.

Optimising Sterilisation Techniques and Callus Induction of Nodes Durio Zibethinus Murr in Vitro Method with Various Media

NASA Astrophysics Data System (ADS)

Hermayani, N.; Retnoningsih, A.; Rahayu, E. S.

2017-04-01

The application of in vitro propagation method needs an aseptic or sterile condition. The objective of the study was to get an optimal sterilisation techniques and medium of callus induction of Durio zibethinus. Sterilisation treatments studied were NaClO and Ca(ClO)2. The three kind of callus induction medium studied were Gamborg (B5), Woody Plant Medium (WPM), and Murashige and Skoog (MS) with the addition of auxin and cytokinin. The experiment unit was three bottles with nodes as explant. Cultures were kept in the culture room for 16 hours daily by LED light intensity of 1000 lux. Parameters investigated of sterilisation technique development was the percentage of contamination, the percentage of explant browning, and the percentage of life explants; whereas the callus induction measured were the rate of callus formation, percentage of callus covered, callus texture, colour and diameter of callus. The results showed that the code of C3 is soaking in calcium hypochlorite (Ca(ClO)2) 50% for 3 minutes, calcium hypochlorite 40% for 2 minutes, and 70% alcohol for 30 seconds separately were suitable for sterilisation of nodes explant. The development of callus on B5 medium with the addition of auxin (2,4-D) 2 ppm and cytokinin (Thidiazuron) 1 ppm was best compared to the other.

Shear Stress-Normal Stress (Pressure) Ratio Decides Forming Callus in Patients with Diabetic Neuropathy

PubMed Central

Noguchi, Hiroshi; Takehara, Kimie; Ohashi, Yumiko; Suzuki, Ryo; Yamauchi, Toshimasa; Kadowaki, Takashi; Sanada, Hiromi

2016-01-01

Aim. Callus is a risk factor, leading to severe diabetic foot ulcer; thus, prevention of callus formation is important. However, normal stress (pressure) and shear stress associated with callus have not been clarified. Additionally, as new valuables, a shear stress-normal stress (pressure) ratio (SPR) was examined. The purpose was to clarify the external force associated with callus formation in patients with diabetic neuropathy. Methods. The external force of the 1st, 2nd, and 5th metatarsal head (MTH) as callus predilection regions was measured. The SPR was calculated by dividing shear stress by normal stress (pressure), concretely, peak values (SPR-p) and time integral values (SPR-i). The optimal cut-off point was determined. Results. Callus formation region of the 1st and 2nd MTH had high SPR-i rather than noncallus formation region. The cut-off value of the 1st MTH was 0.60 and the 2nd MTH was 0.50. For the 5th MTH, variables pertaining to the external forces could not be determined to be indicators of callus formation because of low accuracy. Conclusions. The callus formation cut-off values of the 1st and 2nd MTH were clarified. In the future, it will be necessary to confirm the effect of using appropriate footwear and gait training on lowering SPR-i. PMID:28050567

A Device for Comparing Callus Growth Rates in Vitro

PubMed Central

Krul, William R.; Combs, Michael

1975-01-01

A device to compare the kinetics of callus growth in vitro is described. Changes in volumes of callus grown in scintillation vials were monitored photometrically without removing the sample from the solid support and medium. It is shown that a fiberglass-paper solid support is superior to a plastic foam solid support for the growth of American chestnut callus. PMID:16659126

High Concentration of Benzyladenine Solution Stimulates Anthers for Inducing Callus in Ricinus Communis L.

NASA Astrophysics Data System (ADS)

Liu, Ying; Yan, Shuying; Yang, Fuguang; Li, Dongliang; Tang, Jianian; Liu, Guoxuan; Lin, Shiwan; Niu, Sufang; Yang, Yali

2017-12-01

An high-frequency protocol for induction of callus from anther explants of Ricinus communis was described. When anther explants of R. communis was cultured directly onto medium containing 6-benzylaminopurine (BA) induced formation of only poor quality callus that had a low induction frequency of anther callus (10.67%). However, treating the anther explants with high concentrations (7.5-120 mg/L) of BA solution for short time periods (5-80 min) helped to improve the induction frequency and enhance the quality of the callus formation significantly. The best callus induction (41.25%) was observed when anther explants were treated with 15 mg/L BA solution for 10 min before being inoculated onto hormone-free Murashige and Skoog (MS) medium for 30 days. In order to further optimize the culture system, after treated with 15 mg/L BA for 10 min, anther explants were inoculated on the hormone-free MS medium contained concentrations of sodium nitroprusside (SNP). The results showed that SNP significantly promoted the response of callus induction, especially when 8 mg/L SNP was applied, the the highest percentage of callus induction (60.37%) were gained.

Impact of exogenous ascorbic acid on biochemical activities of rice callus treated with salt stress

NASA Astrophysics Data System (ADS)

Alhasnawi, Arshad Naji; Zain, Che Radziah Che Mohd; Kadhimi, Ahsan A.; Isahak, Anizan; Mohamad, Azhar; Ashraf, Mehdi Farshad; Doni, Febri; Yusoff, Wan Mohtar Wan

2016-11-01

The application of in vitro systems can lead to new methods of crop amelioration. This method has been widely utilized for breeding tenacities, particularly for stress tolerance selection. Salinity causes oxidative stress in callus by enhancing the production of Reactive Oxygen Species (ROS), resulting in an efficient antioxidant system. The exogenous application of ascorbic acid (AsA) is an important requirement for tolerance. The present study aimed to examine in vitro selection strategy for callus induction in rice mature embryo culture on MS culture medium and to produce salt-tolerant callus under sodium chloride (NaCl) and AsA conditions in callus rice variety, MR269. This study also highlights changes in the activities of proline and antioxidants peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) of callus under NaCl stress to understand their possible role in salt tolerance. However, various levels of exogenously applied AsA under saline conditions improved callus, and the antioxidant enzyme activities of AsA are related to resistance to oxidative stress. Our results provide strong support for the hypothesis that AsA-dependent antioxidant enzymes play a significant role in the salinity tolerance of callus rice.

Effect of an ozone injury retardant chemical on isozyme profiles from alfalfa callus in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rier, J.P. Jr.; Sood, V.K.; Whitaker, A.

1983-01-01

Plant ozone injury retardant N-(2-(2-oxo-1-imidazolidinyl)-ethyl)-N'-phenylurea (EDU or ethylenediurea) at 1.0 ppm inhibited growth of callus of alfalfa cultivars Williamsburg (ozone-sensitive) and MSB-CW5An2 (ozone-insensitive) germplasm of Medicago sativa. The presence of EDU (0.1 ppm)in the growth medium increased the number of protein and peroxidase isozyme bands in alfalfa cultivar Williamsburg stem callus and ozone modified their intensities. Protein profiles of MSB stem callus from media containing EDU or exposed to ozone were unchanged. Marked differences were observed between the peroxidase profiles of ozonated and control ozone-insensitive stem callus from media containing EDU. Protein profiles of ozonated ozone-sensitive leaf callus differed slightlymore » from controls. The peroxidase profile of ozonated ozone-sensitive leaf callus was not altered when its growth medium contained EDU, but when it was absent, changes were observed in these profiles.« less

In vitro callus and in vivo leaf extract of Gymnema sylvestre stimulate β-cells regeneration and anti-diabetic activity in Wistar rats.

PubMed

Ahmed, A Bakrudeen Ali; Rao, A S; Rao, M V

2010-11-01

A methanol extract of Gymnema sylvestre leaf and callus showed anti-diabetic activities through regenerating β-cells. Optimum callus was developed under stress conditions of blue light with 2,4-D (1.5 mg/l) and KN (0.5 mg/l), which induced maximum biomass of green compact callus at 45 days, as determined by growth curve analysis. Leaf and optimum callus extracts contains gymnemic acid, which was analyzed using TLC, HPTLC and HPLC methods. The research reported here deals with leaf and callus extracts of G. sylvestre, which significantly increase the weight of the whole body, liver, pancreas and liver glycogen content in alloxan-induced diabetic rats (Wistar rats). The gymnemic acid of leaf and callus extracts significantly increases the regeneration of β-cells in treated rats, when compared with the standard diabetic rats. It could have potential as a pharmaceutical drug for insulin-dependent diabetes mellitus (IDDM). Copyright © 2010 Elsevier GmbH. All rights reserved.

Ca 45 Uptake in Fracture Callus of Normal and Aminoacetonitrile-Treated Rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bolognani, L.; Ponseti, T. V.

1962-04-01

Calcium content and Ca 45 uptake were measured in the fracture callus of normal and AAN-treated rats. It appears that total calcium deposition and Ca 45 uptake are both higher in the young callus, 5 and 10 days after fracture, of the AAN-treated animals. By the 20th day, mineralization of the callus in both groups is similar.

Motion Predicts Clinical Callus Formation

PubMed Central

Elkins, Jacob; Marsh, J. Lawrence; Lujan, Trevor; Peindl, Richard; Kellam, James; Anderson, Donald D.; Lack, William

2016-01-01

Background: Mechanotransduction is theorized to influence fracture-healing, but optimal fracture-site motion is poorly defined. We hypothesized that three-dimensional (3-D) fracture-site motion as estimated by finite element (FE) analysis would influence callus formation for a clinical series of supracondylar femoral fractures treated with locking-plate fixation. Methods: Construct-specific FE modeling simulated 3-D fracture-site motion for sixty-six supracondylar femoral fractures (OTA/AO classification of 33A or 33C) treated at a single institution. Construct stiffness and directional motion through the fracture were investigated to assess the validity of construct stiffness as a surrogate measure of 3-D motion at the fracture site. Callus formation was assessed radiographically for all patients at six, twelve, and twenty-four weeks postoperatively. Univariate and multivariate linear regression analyses examined the effects of longitudinal motion, shear (transverse motion), open fracture, smoking, and diabetes on callus formation. Construct types were compared to determine whether their 3-D motion profile was associated with callus formation. Results: Shear disproportionately increased relative to longitudinal motion with increasing bridge span, which was not predicted by our assessment of construct stiffness alone. Callus formation was not associated with open fracture, smoking, or diabetes at six, twelve, or twenty-four weeks. However, callus formation was associated with 3-D fracture-site motion at twelve and twenty-four weeks. Longitudinal motion promoted callus formation at twelve and twenty-four weeks (p = 0.017 for both). Shear inhibited callus formation at twelve and twenty-four weeks (p = 0.017 and p = 0.022, respectively). Titanium constructs with a short bridge span demonstrated greater longitudinal motion with less shear than did the other constructs, and this was associated with greater callus formation (p < 0.001). Conclusions: In this study of supracondylar femoral fractures treated with locking-plate fixation, longitudinal motion promoted callus formation, while shear inhibited callus formation. Construct stiffness was found to be a poor surrogate of fracture-site motion. Future implant design and operative fixation strategies should seek to optimize 3-D fracture-site motion rather than rely on surrogate measures such as axial stiffness. PMID:26888675

Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae).

PubMed

Chen, J -T.; Chang, W -C.

2000-12-07

An efficient method was established for high frequency somatic embryogenesis and plant regeneration from callus cultures of a hybrid of sympodial orchid (Oncidium 'Gower Ramsey'). Compact and yellow-white embryogenic calli formed from root tips and cut ends of stem and leaf segments on 1/2 MS [11] basal medium supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ, 0.1-3 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 3-10 mg/l) and peptone (1 g/l) for 4-7 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2-4 times (fresh weight) in 1 month. Initiation of somatic embryogenesis and development up to the protocorm-like-bodies (PLBs) from callus cultures was achieved on hormone-free basal medium. Regenerants were recovered from somatic embryos (SEs) after transfer to the same medium and showed normal development. The optimized protocol required about 12-14 weeks from the initiation of callus to the plantlet formation. Generally, the frequency of embryo formation of root-derived callus was higher than stem- and leaf-derived calli. Combinations of naphthaleneacetic acid (NAA) and TDZ significantly promoted embryo formation from callus cultures. The high-frequency (93.8%) somatic embryogenesis and an average of 29.1 SEs per callus (3x3 mm(2)) was found in root-derived callus on a basal medium supplemented with 0.1 mg/l NAA and 3 mg/l TDZ. Almost all the SEs converted and the plantlets grew well with an almost 100% survival rate when potted in sphagnum moss and acclimatized in the greenhouse.

Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.).

PubMed

Templeton-Somers, K M; Collins, W W

1986-03-01

A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in ≦ 11% of the cultures of the total population.

Proline accumulation and its implication in cold tolerance of regenerable maize callus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duncan, D.R.; Widholm, J.M.

1987-03-01

Embryogenic callus of maize (Zea mays L.) inbreds B37wx, H99, H99/sup 3/H95, Mo17, and Pa91 accumulated proline to levels 2.1 to 2.5 times that of control callus when subjected to mannitol-induced water stress, cool temperatures (19/sup 0/C) and abscisic acid (ABA). A combination of 0.53 molar mannitol plus 0.1 millimolar ABA induced a proline accumulation to about 4.5 times that of control callus, equivalent to approximately 0.18 millimoles proline per gram fresh weight of callus. Proline accumulation was directly related to the level of mannitol in the medium. Levels of ABA greater than 1.0 micromolar were required in the mediummore » to induce proline accumulation comparable to that induced by mannitol. Mannitol and ABA levels that induced maximum accumulation of proline also inhibited callus growth and increased tolerance to cold. Proline (12 millimolar) added to culture media also increased the tolerance of callus to 4/sup 0/C. The increased cold tolerance induced by the combination of mannitol and ABA has permitted the storage of the maize inbreds A632, A634Ht, B37wx, C103DTrf, Fr27rhm, H99, Pa91, Va35, and W117Ht at 4/sup 0/C for 90 days which is more than double the typical survival time of callus. These studies show that proline accumulation increase the cold tolerance of regenerable maize callus.« less

[Induction, regeneration, and biolistic sensitivity of different genotypes of common wheat (Triticum aestivum L.)].

PubMed

Fadeev, V S; Shimshilashvili, Kh R; Gaponenko, A K

2008-09-01

The induction, regeneration, and biolistic sensitivities of different genotypes of common wheat (Triticum aestivum L.) have been determined in order to develop an efficient system for transformation of Russian cultivars of spring wheat. Short-term (two days) cold treatment (4 degrees C) has been demonstrated to distinctly increase the frequency of morphogenetic callus induction. The optimal phytohormonal composition of the nutrient medium ensuring an in vitro regeneration rate of the common wheat cultivar Lada as high as 90% has been determined. The optimal temporal parameters of genetic transformation of wheat plants (10-14 days of culturing after initiation of a morphogenetic callus) have been determined for two transformation methods: biolistic without precipitated DNA and transformation with the plasmid psGFP-BAR. Analysis of the transient expression of the gfp gene has confirmed that 14 days of culturing is the optimal duration.

Effect of Antioxidants and Carbohydrates in Callus Cultures of Taxus brevifolia: Evaluation of Browning, Callus Growth, Total Phenolics and Paclitaxel Production

PubMed Central

Yari Khosroushahi, Ahmad; Naderi-Manesh, Hossein; Toft Simonsen, Henrik

2011-01-01

Introduction To control the tissue browning phenomenon, callus growth, total phenolics and paclitaxel production, in the current investigation, we evaluated the effects of citric acid and ascorbic acid (as antioxidants) and glucose, fructose and sucrose in callus cultures of Taxus brevifolia. Methods To obtain healthy callus/cell lines of Taxus brevifolia, the effects of two antioxidants ascorbic acid (100-1000 mg/L) and citric acid (50-500 mg/L), and three carbohydrates (glucose, fructose and sucrose (5-10 g/L)) were studied evaluating activities of polyphenol oxidase (PPO) and peroxidase (PO) enzymes, callus growth/browning, total phenolics and paclitaxel production. Results These antioxidants (ascorbic acid and citric acid) failed to show significant effects on callus growth, browning intensity or paclitaxel production. However, the carbohydrates imposed significant effects on the parameters studied. High concentrations of both glucose and sucrose increased the browning intensity, thus decreased callus growth. Glucose increased paclitaxel production, but sucrose decreased it. Conclusion These results revealed that the browning phenomenon can be controlled through supplementation of the growth media with glucose, sucrose (5 g/L) and fructose (10 g/L), while increased paclitaxel production can be obtain by the optimized media supplemented with glucose (10 g/L), sucrose and fructose (5 g/L). PMID:23678406

Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. [Nicotiana tabacum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Constantin, M.J.; Henke, R.R.; Mansur, M.A.

1977-01-01

Incorporating activated charcoal (AC) in culture media has been shown to affect growth and development of various organisms. Since AC stimulates the development of tobacco haploid plantlets from cultured anthers, research was conducted to determine the effect of activated charcoal on pith-derived callus growth and shoot development in Nicotiana tabacum cv. Wisconsin 38. Our results indicate that the hormones required for callus growth and shoot development in Wisconsin-38 tobacco are adsorbed by AC, thereby inhibiting callus growth and prohibiting shoot development. This effect was observed even when AC was removed from the medium by filtration prior to culturing the callus.

In Vitro Culture and Phytochemical Analysis of Passiflora tenuifila Killip and Passiflora setacea DC (Passifloraceae).

PubMed

Sozo, Jenny Sumara; Cruz, Daniel Cuzziol; Pavei, Ana Flavia; Pereira, Isadora Medeiros da Costa; Wolfart, Marcia; Ramlov, Fernanda; Fiuza Montagner, Daiane; Maraschin, Marcelo; Viana, Ana Maria

2016-01-01

We have developed reproducible micropropagation, callus culture, phytochemical, and antioxidant analysis protocols for the wild passion fruit species P. tenuifila, and P. setacea, native to the Brazilian endangered biomes Atlantic Forest, Cerrado, and Caatinga, by using seeds and explants from seedlings and adult plants. Genotype and explant origin-linked differences are visible amongst the Passiflora species concerning callus production, total phenolics, and antioxidant activity. The protocols developed for screening phytochemicals and antioxidants in P. tenuifila and P. setacea callus extracts have shown their potential for phenolic production and antioxidant activity. The high level of phenolic compounds seems to account for the antioxidant activity of methanolic extracts of P. tenuifila derived from 45-day-old immature seed callus. The methanolic extracts of callus derived from P. setacea seedling leaf node and cotyledonary node explants have shown the highest antioxidant activity despite their lower content of phenolics, as compared to cotyledon callus extracts. The optimized micropropagation and callus culture protocols have great potential to use cell culture techniques for further vegetative propagation, in vitro germplasm conservation, and secondary metabolite production using biotic and abiotic elicitors.

Flavonol dimers from callus cultures of Dysosma versipellis and their in vitro neuraminidase inhibitory activities.

PubMed

Chen, Ridao; Duan, Ruigang; Wei, Yannan; Zou, Jianhua; Li, Junwei; Liu, Xiaoyue; Wang, Haiyan; Guo, Ying; Li, Qiuhong; Dai, Jungui

2015-12-01

A chemical investigation of callus cultures of Dysosma versipellis led to the isolation of five new flavonol dimers, dysoverines A-E (1-5), together with 12 known compounds (6-17). The structures of new compounds were determined by the extensive spectroscopic data analyses. The biosynthetic pathway of the new compounds was proposed to involve O-methylation, prenylation, and Diels-Alder cycloaddition, which successively occurred in cultured plant cells. Compounds 1-17 exhibited in vitro neuraminidase inhibitory activities with the IC50 values of 31.0-93.9μM. Copyright © 2015 Elsevier B.V. All rights reserved.

A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

PubMed

Murata, M; Nishimura, M; Murai, N; Haruta, M; Homma, S; Itoh, Y

2001-02-01

Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

PubMed

Rygula, Anna; Oleszkiewicz, Tomasz; Grzebelus, Ewa; Pacia, Marta Z; Baranska, Malgorzata; Baranski, Rafal

2018-05-15

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of β-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells. Copyright © 2018 Elsevier B.V. All rights reserved.

The Influence of Physical Forces on Progenitor Cell Migration, Proliferation and Differentiation in Fracture Repair

DTIC Science & Technology

2007-11-01

accelerated healing patterns in the loaded specimens. Periosteal callus formation appears more robust with more chondrocytes present in loaded... periosteal callus formation on one side of the fracture gap. It is hypothesized that callus development may occur first on the medial side of the femoral...Figure 10: Comparison of periosteal callus formation (trichrome stain) between a loaded limb at section levels 1 (a), 3 (b), and 5 (c), and

ADSORPTION OF PHOSPHOROUS BY CATTAIL CALLUS CELLS

EPA Science Inventory

Data from this study demonstrates that cattail callus cells can be used to predict the phosphorus concentration in cattail leaves when they are supplied with similar phosphorus levels. If this relationship between callus cells and whole plants is found applicable to other marsh p...

Callus Induction from Various Organs of Dragon Fruit, Apple and Tomato on some Mediums.

PubMed

Rumiyati; Sismindari; Semiarti, Endang; Milasari, Asri Fajar; Sari, Dheatika Karina; Fitriana, Nia; Galuh, Sekar

2017-01-01

Dragon fruit (Hylocereus spp.), apple (Malus sylvestris Mill.) and tomato (Solanum lycopersicum L.) are high potential sources of antioxidant compounds such as phenolics. The compounds have the capability of protecting cells and tissues against free radicals. Secondary metabolite produced by callus cell culture from plant organs also acts as a source of antioxidants. This study aimed to determine the optimal ratio of sucrose and 2,4-D in Murashige and Skoog (MS) medium for callus induction from different plant organ explants. With all of characteristic, callus can be used further for the development of natural cell regeneration agent. This study was conducted using analytical technique. Suitable explants were obtained. They were developed in various concentrations of combination between MS medium and 2,4-D. Callus growth, including their weight and surface was then measured and analyzed by using one-way analysis of variance (ANOVA). Callus was able to grow from its explants in 5-7 days after induction process. They were clear in color and had friable texture. The highest value of fresh weight of dragon fruit callus was obtained through MS supplemented with 1 μL L-1 2,4-D and 30 g sucrose. However, apple and tomato callus induction and growth maintenance reached optimal medium on MS supplemented with 30 g sucrose and 2 μL L-1 2,4-D. Callus of apple, dragon fruit and tomato was maintained upon MS supplemented with 30-40 g sucrose and 1-2 μL L-1 2,4-D for optimum induction and growth. The optimization of growth medium will give advantages for further development of natural cell regeneration agent.

Genetic Dissection of Maize Embryonic Callus Regenerative Capacity Using Multi-Locus Genome-Wide Association Studies

PubMed Central

Ma, Langlang; Liu, Min; Yan, Yuanyuan; Qing, Chunyan; Zhang, Xiaoling; Zhang, Yanling; Long, Yun; Wang, Lei; Pan, Lang; Zou, Chaoying; Li, Zhaoling; Wang, Yanli; Peng, Huanwei; Pan, Guangtang; Jiang, Zhou; Shen, Yaou

2018-01-01

The regenerative capacity of the embryonic callus, a complex quantitative trait, is one of the main limiting factors for maize transformation. This trait was decomposed into five traits, namely, green callus rate (GCR), callus differentiating rate (CDR), callus plantlet number (CPN), callus rooting rate (CRR), and callus browning rate (CBR). To dissect the genetic foundation of maize transformation, in this study multi-locus genome-wide association studies (GWAS) for the five traits were performed in a population of 144 inbred lines genotyped with 43,427 SNPs. Using the phenotypic values in three environments and best linear unbiased prediction (BLUP) values, as a result, a total of 127, 56, 160, and 130 significant quantitative trait nucleotides (QTNs) were identified by mrMLM, FASTmrEMMA, ISIS EM-BLASSO, and pLARmEB, respectively. Of these QTNs, 63 QTNs were commonly detected, including 15 across multiple environments and 58 across multiple methods. Allele distribution analysis showed that the proportion of superior alleles for 36 QTNs was <50% in 31 elite inbred lines. Meanwhile, these superior alleles had obviously additive effect on the regenerative capacity. This indicates that the regenerative capacity-related traits can be improved by proper integration of the superior alleles using marker-assisted selection. Moreover, a total of 40 candidate genes were found based on these common QTNs. Some annotated genes were previously reported to relate with auxin transport, cell fate, seed germination, or embryo development, especially, GRMZM2G108933 (WOX2) was found to promote maize transgenic embryonic callus regeneration. These identified candidate genes will contribute to a further understanding of the genetic foundation of maize embryonic callus regeneration. PMID:29755499

Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells.

PubMed

Iwai, H; Ishii, T; Satoh, S

2001-10-01

When leaf disks from haploid plants of Nicotiana plumbaginifolia Viv. were transformed with T-DNA and cultured on shoot-inducing medium, nonorganogenic callus. designated nolac (for non-organogenic callus with loosely attached cells), appeared on approximately 7% of leaf disks. In contrast, normal callus was generated on T-DNA-transformed leaf disks from diploid plants and on non-transformed leaf disks from haploid and diploid plants. Transmission electron microscopy revealed that the middle lamellae and the cell walls of one line of mutant callus (nolac-H14) were barely stained by ruthenium red. even after demethylesterification with NaOH, whereas the entire cell wall and the middle lamella were strongly stained in normal callus. In cultures of nolac-H14 callus, the level of sugar components of pectic polysaccharides in the hemicellulose fraction was reduced and that in the culture medium was elevated, as compared with cultures of normal callus. These results indicate that pectic polysaccharides are not retained in the cell walls and middle lamellae of nolac-H14 callus. In nolac-H14, the ratio of arabinose to galactose was low in the pectic polysaccharides purified from all cell wall fractions and from the medium, in particular, in the hemicellulose fractions. The low levels of arabinofuranosyl (T-Araf, 5-Araf, 2,5-Araf, and 3,5-Araf) residues in the pectic polysaccharides of the hemicellulosic fraction of nolac-H,14 indicated that no neutral-sugar side chains, composed mainly of linear arabinan. were present in nolac-H14. Arabinose-rich pectins. which are strongly associated with cellulose-hemicellulose complexes, might play an important role in intercellular attachment in the architecture of the cell wall.

Effects of hypoxia condition in embryogenic callus growth of soybean cell culture

NASA Astrophysics Data System (ADS)

Damanik, R. I.; Manurung, B. H.; Bayu, E. S.

2018-02-01

The study was performed at Tissue Culture Laboratory, Agrotechnology Department, University of Sumatera Utara, to investigate the effect of plant growth regulator (PGR) and embryogenic callus performance soybean cultivars on hypoxia condition. This research had two stages, induction of embryogenic callus and analysis metabolism of callus after hypoxic condition with T-test. The analysis was used factorial Completely Randomized Design with two factors. The first factors were cultivars of soybean (Baluran, Gepak Kuning, and Grobogan) and the second factors were combinations of PGR (5 mg/l 2,4-D + 1 mg/l BAP, 10 mg/l 2,4-D + 1.5 mg/l BAP, and 15 mg/l 2,4-D + 2 mg/l BAP). The result showed the cultivars, combination of PGR, and interaction between cultivars and PGR gave significant effect to weight callus. The result of T-test showed that in hypoxic condition, POD enzyme exercise on Gepak Kuning’s callus in 5 mg/l 2,4-D + 1 mg/l BAP was different before and after hypoxic condition.

Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus.

PubMed

Gomez, M P; Segura, J

1996-08-01

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.

Regeneration of Cuphea wrightii (Peyr 651) and fertile C.wrightii C. tolucana hybrids from leaf explants.

PubMed

Przybecki, Z; Olejniczak, J; Adamska, E

2001-01-01

Callus was obtained from leaf explants of Cupea wrightii and Cuphea wrightii x Cuphea tolucana hybrid plants, and the plants were later regenerated. C. tolucana explants were capable of forming callus, but not of regenerating. In order to obtain callus from C. wrightii and the hybrid plants, the addition of BAP to the medium was necessary, whereas in the case of C. tolucana auxin was needed. The regeneration of the plants did not require auxin, and both forms (C. wrightii and the hybrids) regenerated in the same medium. The regeneration yield came to around 12 plants from the callus of one harvest. Some of the callus from the hybrids was subjected to colchicine treatment, which increased the number of fully fertile regenerants from 1% to almost 20%.

Callus Growth Kinetics of Physic Nut (Jatropha curcas L.) and Content of Fatty Acids from Crude Oil Obtained In Vitro.

PubMed

da Luz Costa, Jefferson; da Silva, André Luís Lopes; Bier, Mário César Jucoski; Brondani, Gilvano Ebling; Gollo, André Luiz; Letti, Luiz Alberto Junior; Erasmo, Eduardo Andrea Lemus; Soccol, Carlos Ricardo

2015-06-01

The callus growth kinetics allows identifying the appropriate moment for callus pealing and monitoring the accumulation of primary and secondary metabolites. The physic nut (Jatropha curcas L.) is a plant species used for biofuel production due to its high oil content; however, this plant presents a great amount of bioactive compounds which can be useful for industry. The aim of this research was to establish a calli growth curve and to evaluate the fatty acid profile of crude oil extracted from callus. The callus growth kinetics presented a sigmoid standard curve with six distinct phases: lag, exponential, linear, deceleration, stationary, and decline. Total soluble sugars were higher at the inoculation day. Reducing sugars were higher at the inoculation day and at the 80th day. The highest percentage of ethereal extract (oil content) was obtained at the 120th day of culture, reaching 18 % of crude oil from the callus. The calli produced medium-chain and long-chain fatty acids (from 10 to 18 carbon atoms). The palmitic acid was the fatty acid with the highest proportion in oil (55.4 %). The lipid profile obtained in callus oil was different from the seed oil profile.

Isozyme modifications and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas (L.) Lam.).

PubMed

Cavalcante Alves, J M; Sihachakr, D; Allot, M; Tizroutine, S; Mussio, I; Servaes, A; Ducreux, G

1994-05-01

The potential of somatic embryogenesis was evaluated for 10 cultivars of sweet potato through extensive embryogenic response and isozyme analysis. Embryogenic callus was induced by incubating lateral buds on Murashige and Skoog medium containing 10 μM 2,4-dichlorophenoxyacetic acid for 6-8 weeks. The frequency of embryogenic response was low, and varied with genotypes, ranging from 0 to 17%. Embryo to plantlet formation could be enhanced by the use of the combination of 2,4-dichlorophenoxyacetic acid with kinetin, both used at 0.01 μM. Embryogenic callus with its potential of plantlet formation has constantly been maintained for over two years. However, after several subcultures, 0.5 to 12% of embryogenic callus reverted irreversibly into friable fast-growing non-embryogenic callus whose ability to regenerate shoots was then definitively lost. The isozymes of esterase, peroxidase, glutamate oxaloacetate transaminase and acid phosphatase investigated in this study were found appropriate to distinguish compact embryogenic from friable non-embryogenic callus in sweet potato. In fact, the callus reversion was associated with a loss of bands or a decline in isozyme activity. On the contrary, very small changes in isozyme activity or no specific changes at all were observed during the differentiation of embryogenic callus into globular embryos.

Embryogenic competence acquisition in sugarcane callus is associated with differential H+ pump abundance and activity.

PubMed

Passamani, Lucas Z; Bertolazi, Amanda A; Ramos, Alessandro C; Santa-Catarina, Claudete; Thelen, Jay J; Silveira, Vanildo

2018-06-22

Somatic embryogenesis is an important biological process in several plant species, including sugarcane. Proteomics approaches have shown that H + pumps are differentially regulated during somatic embryogenesis; however, the relationship between H + flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H + flux and somatic embryo maturation in sugarcane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H + flux and H + pump (P-H + -ATPase, V-H + -ATPase and H + -PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H + pumps. We observed that P-H + -ATPase and H + -PPase were more abundant in embryogenic callus. Compared with non-embryogenic callus, embryogenic callus showed higher H + influx, especially on maturation day 14, as well as higher H+ pump activity (mainly P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H + pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H + pump protein abundance and, consequently, higher H + flux and embryogenic competence acquisition in the callus of sugarcane, allowing for optimization of the somatic embryo conversion process by modulating the activities of these H + pumps.

Influence of light quality on growth, secondary metabolites production and antioxidant activity in callus culture of Rhodiola imbricata Edgew.

PubMed

Kapoor, Sahil; Raghuvanshi, Rinky; Bhardwaj, Pushpender; Sood, Hemant; Saxena, Shweta; Chaurasia, Om Prakash

2018-06-01

Rhodiola imbricata is a rare medicinal herb well-known for its adaptogenic and antioxidant properties due to the presence of a diverse array of secondary metabolites, including phenylethanoids and phenylpropanoids. These secondary metabolites are generating considerable interest due to their potential applications in pharmaceutical and nutraceutical industries. The present study investigated the influence of light quality on growth, production of industrially important secondary metabolites and antioxidant activity in callus cultures of Rhodiola imbricata. Callus cultures of Rhodiola imbricata were established under different light conditions: 100% red, 100% blue, 100% green, RGB (40% red: 40% green: 20% blue) and 100% white (control). The results showed that the callus cultures grown under red light accumulated maximum amount of biomass (7.43 g/l) on day 21 of culture, as compared to other light conditions. Maximum specific growth rate (0.126 days -1 ) and doubling time (132.66 h) was observed in callus cultures grown under red light. Reverse phase-high performance liquid chromatographic (RP-HPLC) analysis revealed that the callus cultures exposed to blue light accumulated maximum amount of Salidroside (3.12 mg/g DW) on day 21 of culture, as compared to other light conditions. UV-Vis spectrophotometric analysis showed that the callus cultures exposed to blue light accumulated maximum amount of total phenolics (11.84 mg CHA/g DW) and total flavonoids (5.53 mg RE/g DW), as compared to other light conditions. Additionally, callus cultures grown under blue light displayed enhanced DPPH free radical scavenging activity (53.50%). Callus cultures grown under different light conditions showed no significant difference in ascorbic acid content (11.05-13.90 mg/g DW) and total antioxidant capacity (27.37-30.17 mg QE/g DW). The correlation analysis showed a positive correlation between total phenolic content and DPPH free radical scavenging activity in callus cultures (r = 0.85). Taken together, these results demonstrate the remarkable potential of light quality on biomass accumulation and production of industrially important secondary metabolites in callus cultures of Rhodiola imbricata. This study will open new avenues and perspectives towards abiotic elicitation strategies for sustainable growth and enhanced production of bioactive compounds in in-vitro cultures of Rhodiola imbricata. Copyright © 2018 Elsevier B.V. All rights reserved.

Mevalonate kinase activity during different stages of plant regeneration from nodular callus cultures in white pine (Pinus strobus).

PubMed

Tang, Wei; Newton, Ronald J

2006-02-01

Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting. Nodular calli were induced from Pinus strobus (PS) embryos by culture in nodular callus induction medium in a 0-, 8- or 16-h photoperiod. Mevalonate kinase activity peaked in nodular calli after three weeks of culture on nodular callus induction medium in a 16-h photoperiod, whereas frequency of nodular callus formation peaked after 4 weeks of culture on nodular callus induction medium in darkness. During adventitious shoot formation, MK activity peaked in shoots derived from dark-grown nodular calli after 3 weeks on bud formation medium, and frequency of shoot formation was highest in dark-grown nodular calli cultured on bud formation medium for 4 weeks. During rooting, MK activity peaked 2 weeks after transfer of adventitious shoots to rooting medium and rooting frequency was highest in adventitious shoots after 3 weeks on rooting medium. Although during nodular callus induction in darkness MK activity was inversely related to frequency of nodular callus formation, MK activity was highly correlated with frequency of shoot formation and with rooting frequency. The observed increase in MK activity preceding rooting suggests that MK could serve as a marker for rooting of white pine shoots in vitro.

Phytochemical and Bioactive Potential of in vivo and in vitro Grown Plants of Centaurea ragusina L. - Detection of DNA/RNA Active Compounds in Plant Extracts via Thermal Denaturation and Circular Dichroism.

PubMed

Vujčić, Valerija; Radić Brkanac, Sandra; Radojčić Redovniković, Ivana; Ivanković, Siniša; Stojković, Ranko; Žilić, Irena; Radić Stojković, Marijana

2017-11-01

The phytochemical composition and biological activity of non-volatile components of Centaurea ragusina L. has not been studied previously. Our aim was to evaluate the phytochemical and bioactive potential (including interactions with polynucleotides) of C. ragusina L. depending on the origin of plant material (in vivo - leaves from natural habitats, ex vitro - leaves from plants acclimated from culture media, in vitro - leaves and calli from plants grown in culture media) and polarity of solvents used in extract preparation (80 and 96% ethanol and water combinations or single solvents). The polyphenol composition was determined by spectrophotometric and HPLC analysis. Biological activity of extracts was evaluated by following methods: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods for antioxidative activity, 2,3,5-triphenyl tetrazolium chloride (TTC) microdilution method for antibacterial activity, crystal-violet test for cytotoxic activity and thermal denaturation (TD) and circular dichroism (CD) for DNA/RNA interactions. Conditions for the most efficient polyphenol extraction were determined: the 80% ethanol/water solvent system was the most suitable for callus and leaf ex vitro samples and 80 or 96% ethanol for leaf in vivo samples. Significantly higher levels of chlorogenic acid and naringenin were detected in callus tissue than in vivo plant. Ethanolic extracts exhibited the significant antibacterial activity against Staphylococcus aureus ATCC 25923. DNA/RNA active compounds in plant extracts were detected by TD and CD methods. Callus tissue and ex vitro leaves represent a valuable source of polyphenols as in vivo leaves. TD and CD can be applied for detection of DNA/RNA active compounds in extracts from natural resources. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Shikonin Production by Callus Culture of Onosma bulbotrichom as Active Pharmaceutical Ingredient

PubMed Central

Bagheri, Fereshteh; Tahvilian, Reza; Karimi, Naser; Chalabi, Maryam; Azami, Mahsa

2018-01-01

The objective of this research was in-vitro germination and callus induction of Onosma bulbotrichum (O. bulbotrichum) as a medicinal herb which belongs to Boraginaceae family. For germination, the seeds were cultured on growth regulator-free MS medium and for callus induction, seeds were sown on modified MS medium containing different concentrations of kinetin (kn)- Indole-3-acetic acid (IAA) and kn- 2,4-D (2,4-dichlorophenoxyacetic acid), respectively. The plates were maintained in the dark at growth chamber. After 7 days seed germination on hormone-free medium and after 10 days callus initiation on modified medium in the presence of hormones was occurred. The maximum pigmented callus (100%) was observed on modified MS medium with a combination of 0.2 mg.L-1 IAA + 2.10 mg.L-1 kn. Shikonin determination was performed by HPLC method. In addition, total hydroxynaphtoquinons as polyphenols in sum of callus and culture medium were measured by spectrophotometric method and revealed that total naphtoquinones content at IAA was more than 2, 4-D. PMID:29881407

Primary characterization and evaluation of anti ulcerogenic activity of an aqueous extract from callus culture of Cereus peruvianus Mill. (Cactaceae).

PubMed

Jayme, Milena O; Ames, Franciele Q; Bersani-Amado, Ciomar A; Machado, Maria de Fatima P S; Mangolin, Claudete A; Goncalves, Regina A C; de Oliveira, Arildo J B

2015-01-01

In the current study we reported cultivation, extraction procedure, analysis and preliminary characterization of the aqueous extract from Cereus peruvianus callus culture and evaluated its anti ulcerogenic activity in vivo models of experimental ulcers in Wistar rats. The obtained aqueous extract from callus (AC) was dialyzed and subjected to freeze-thaw process, providing a possible polysaccharide. The carbohydrate and protein contents of the aqueous extract were estimated at 53.4% and 0.66%, respectively, composed primarily of galactose, arabinose and galacturonic acid, with minor amounts of glucose. This appeared heterogeneous when analyzed by high-performance size-exclusion chromatography and a multiangle laser light scattering detector (HPSEC-MALLS). The AC was found to be significantly effective against ethanol-induced lesions but was ineffective against indomethacin-induced lesions. The callus culture of C. peruvianus is an alternative source for the synthesis of substances originally produced by plants. The calluses grown indefinitely in vitro under controlled conditions are stable tissues, and the aqueous extract from calluses may be used instead of fully developed plants using the protocols described in this study.

The ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing.

PubMed

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla A; Diekwisch, Tom; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBN(Δ5-6) truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

The Ameloblastin extracellular matrix molecule enhances bone fracture resistance and promotes rapid bone fracture healing

PubMed Central

Lu, Xuanyu; Li, Wenjin; Fukumoto, Satoshi; Yamada, Yoshihiko; Evans, Carla; Diekwisch, Thomas G.H.; Luan, Xianghong

2016-01-01

The extracellular matrix (ECM) provides structural support, cell migration anchorage, cell differentiation cues, and fine-tuned cell proliferation signals during all stages of bone fracture healing, including cartilaginous callus formation, callus remodeling, and bony bridging of the fracture gap. In the present study we have defined the role of the extracellular matrix protein ameloblastin (AMBN) in fracture resistance and fracture healing of mouse long bones. To this end, long bones from WT and AMBNΔ5-6 truncation model mice were subjected to biomechanical analysis, fracture healing assays, and stem cell colony formation comparisons. The effect of exogenous AMBN addition to fracture sites was also determined. Our data indicate that lack of a functional AMBN in the bone matrix resulted in 31% decreased femur bone mass and 40% reduced energy to failure. On a cellular level, AMBN function inhibition diminished the proliferative capacity of fracture repair callus cells, as evidenced by a 58% reduction in PCNA and a 40% reduction in Cyclin D1 gene expression, as well as PCNA immunohistochemistry. In terms of fracture healing, AMBN truncation was associated with an enhanced and prolonged chondrogenic phase, resulting in delayed mineralized tissue gene expression and delayed ossification of the fracture repair callus. Underscoring a role of AMBN in fracture healing, there was a 6.9-fold increase in AMBN expression at the fracture site one week after fracture, and distinct AMBN immunolabeling in the fracture gap. Finally, application of exogenous AMBN protein to bone fracture sites accelerated callus formation and bone fracture healing (33% increase in bone volume and 19% increase in bone mineral density), validating the findings of our AMBN loss of function studies. Together, these data demonstrate the functional importance of the AMBN extracellular matrix protein in bone fracture prevention and rapid fracture healing. PMID:26899203

Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

PubMed

Chen, M H; Wang, P J; Maeda, E

1987-10-01

The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

Influence ofArtemisia princeps var.orientalis components on callus induction and growth.

PubMed

Kil, B S; Yun, K W; Lee, S Y

1992-08-01

An in vitro study was performed to determine the potential application of tissue culture in determining allelopathic potentialof Artemisia princeps var.Orientalis (wormwood). Aqueous extracts and volatile substances ofA. princeps var.Orientalis were tested to determine their effects on callus induction and growth of several tested species. Extracts of 5%A. princeps var.Orientalis caused some reduction in concentration, induction, and growth of callus, although they looked normal, whereas the expiants of most receptor plants did not develop callus at higher concentration. Lettuce andEclipta prostrata were the most sensitive species, andA. princeps var.Orientalis was affected by its own extracts. The growth of calluses in MS 121 medium treated with essential oil ofA. princeps var.Orientalis was inhibited, and the degree of inhibition was proportional to the concentration of the essential oil.

Radiation effects on the resting and proliferating cells in normal tissue of mouse (in Japanese)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayashi, S.

1972-10-01

The investigation was planned to compare the radiosensitivity of callus- forming cells in resting phase with that in proliferating phase and to compare the recovery of sublethal damage of callusforming cells in resting phase with that in proliferating phase. Experimental animals were 8-week-old female I.C.R./ J.C.L. mice. The maximum sizes of callus were nearly constant among control mice without irradiation after fracture. They, however, were inhibited with administered doses and seemed to be reflected by the Proliferating ability of callus-forming cells after irradiation. The analysis was performed by C.I.D. 50 (callus inhibition dose 50) or dose that produced a specifiedmore » inhibition of callus size in half of the subjects. The callus-forming cells in adult mice were in resting phase without any stimulations, but they extensively entered into proliferating phase after fracture. The labeling index rose around 6 hrs after fracture and reached 9% of the maximum value at 72 are after fracture. Mice were followed by x-ray projection until 60 days after irradiation to observe the callus sizes, and the maximum sizes of callus for each mouse were examined by planimetry to calculate the C.I.D. 50. The callus-forming cell was more radioresistant in resting phase by a factor of 1.5 to 2.0 than in proliferating phase. The cell in resting phase demonstrated a marked recovery of sublethal damage in 4 hrs after administration of 1.000 rads, and it showed essentially no more changes in recovery with the increased time interval to 24 hrs, while the cell in proliferating phase demonstrated almost full recovery of sublethal damage is 2 hrs after administration of 300 rads and showed a fluctuated pattern of recovery with a dip at 4 hrs of the time interval in two fractions. (auth)« less

Effect of Hypergravity and Phytohormones on Isoflavonoid Accumulation in Soybean ( Glycine max. L.) Callus

NASA Astrophysics Data System (ADS)

Downey, Peter J.; Levine, Lanfang H.; Musgrave, Mary E.; McKeon-Bennett, Michelle; Moane, Siobhán

2013-02-01

The objective of this study was to explore the potential interaction between gravity and growth hormones on isoflavonoid accumulation. Soybean callus ( Glycine max (L.) Merr. cv. `Acme') was grown in the dark for 16 days at 22 °C in a growth medium supplemented with four different combinations of phytohormones and subjected to 4- g and 8- g forces simulated in a centrifuge and 1- g in an adjacent stationary control. Isoflavonoid aglycones and their glycoside concentrations (daidzein, genistein, daidzin, 6″-O-malonyl-7-O-glucosyl daidzein, genistin, 6″-O-malonyl-7-O-glucosyl genistein) were determined in the resulting tissues. Although gravity had no significant impact on callus growth, increasing gravity reduced isoflavonoid accumulation in three out of the four phytohormone-supplemented culture media. The ratio of the auxin naphthalene acetic acid (NAA) to the cytokinin benzylaminopurine (BAP) was found to have profound effect on both callus growth and isoflavonoid accumulation. The cytokinin BAP promoted callus tissue growth, but reduced callus isoflavonoid suggesting the isoflavonoid accumulation was not keeping pace with the cell growth in the elevated concentration of BAP. On the other hand, NAA had little or no effect on callus growth, but greatly enhanced isoflavonoid accumulation. Interactive effects of gravity and hormone on isoflavonoid accumulation were evident and its implication to the mechanism by which gravity exerts the effect on plant secondary metabolites is discussed.

Hot callusing for propagation of American beech by grafting

Treesearch

David W. Carey; Mary E. Mason; Paul Bloese; Jennifer L. Koch

2013-01-01

To increase grafting success rate, a hot callus grafting system was designed and implemented as part of a multiagency collaborative project to manage beech bark disease (BBD) through the establishment of regional BBD-resistant grafted seed orchards. Five years of data from over 2000 hot callus graft attempts were analyzed using a logistic regression model to determine...

Production of chlorogenic acid in Varthemia persica DC (var. persica) callus cultures

PubMed Central

Siahpoush, A.; Ghasemi, N.; Ardakani, M. Shams; Asghari, G.

2011-01-01

Chlorogenic acid, a pharmacologically important compound, is a phenolic compound that occurs in certain commonly used medicinal herbs. We looked for the presence of this compound in the callus cultures of Varthemia persica DC (var. persica). We have evaluated the conditions for establishment of callus cultures of V. persica and the in vitro production of chlorogenic acid. Callus was initiated by culturing seedling of V. persica on MS basal medium supplemented with different concentrations of kinetin, naphthalene acetic acid and 2,4-diphenoxy acetic acid. Also, the influence of light, and phytohormones on the production of chlorogenic acid was examined. Kinetin stimulated the production of chlorogenic acid. Replacement of 2,4-diphenoxy acetic acid with naphthalene acetic acid did not alter the chlorogenic acid production. The ability to induce the accumulation of chlorogenic acid in the V. persica callus cultures offers an opportunity to produce a phenolic compound with therapeutic value. PMID:22049279

Experimental stimulation of bone healing with teriparatide: histomorphometric and microhardness analysis in a mouse model of closed fracture.

PubMed

Mognetti, Barbara; Marino, Silvia; Barberis, Alessandro; Martin, Anne-Sophie Bravo; Bala, Yohann; Di Carlo, Francesco; Boivin, Georges; Barbos, Michele Portigliatti

2011-08-01

Fracture consolidation is a crucial goal to achieve as early as possible, but pharmacological stimulation has been neglected so far. Teriparatide has been considered for this purpose for its anabolic properties. We set up a murine model of closed tibial fracture on which different doses of teriparatide were tested. Closed fracture treatment avoids any bias introduced by surgical manipulations. Teriparatide's effect on callus formation was monitored during the first 4 weeks from fracture. Callus evolution was determined by histomorphometric and microhardness assessment. Daily administration of 40 μg/kg of teriparatide accelerated callus mineralization from day 9 onward without significant increase of sizes, and at day 15 the microhardness properties of treated callus were similar to those of bone tissue. Teriparatide considerably improved callus consolidation in the very early phases of bone healing.

In vivo and in vitro evaluation of sterols from Gymnema sylvestrte R. Br.

PubMed

Vats, Sharad; Kamal, Raka

2013-12-01

Gymnema sylvestre R. Br. is an important medicinal plant known for its antidiabetic potential. In the present study, phytosterols from G. sylvestre was identified and quantified in vivo and in vitro. Maximum callus induction was observed in MS medium supplemented with 0.5 mg L(-1) of 2, 4-D. The protein content was significantly high both in aerial plant parts and callus tissue. Phytosterols were identified using chromatographic and spectral studies. beta-sitosterol, campesterol and stigmasterol were identified both in vivo and in vitro. Lanosterol was identified only in callus culture. Phytosterols have reported for the first time in callus culture of G. sylvestre.

Direct and indirect organogenesis of Alpinia galanga and the phytochemical analysis.

PubMed

Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Gandi, Suryakala; Mangamoori, Lakshmi Narasu; Giri, Archana

2011-11-01

Alpinia galanga is a rhizomatous herb rich in essential oils and various other significant phytoconstituents. Rapid direct regeneration was obtained from the rhizome explants (15.66 ± 0.57 shoots) on MS media supplemented with zeatin at a concentration of 2 mg/l. The callus cultures of A. galanga were initiated from the rhizome explants on MS media supplemented with 2 mg/l each of BAP, 2,4-D, and NAA. The callus was analyzed for the presence of a vital phytoconstituent--acetoxychavicol acetate (ACA) associated with various biological properties. ACA was detected in the young friable callus as well as the stationary phase callus. Moreover, the induction of morphogenetic response in callus resulted in higher accumulation of ACA. The phytohormone withdrawal from the propagation media and the subsequent transfer of callus to BAP (2 mg/l) containing MS media has resulted in multiple shoot induction. The regenerated (indirect) plants have shown 1.6-fold higher ACA content (1.253%) when compared to the control plant (0.783%). Micropropagation of such conventionally propagated plants is very essential to meet the commercial demand as well as to ensure easy storage and transportation of disease free stocks.

In vitro callus culture of Heliotropium indicum Linn. for assessment of total phenolic and flavonoid content and antioxidant activity.

PubMed

Kumar, Muthusamy Senthil; Chaudhury, Shibani; Balachandran, Srinivasan

2014-12-01

The total phenolic and flavonoid content and percentage of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of callus and in vivo plant parts of Heliotropium indicum Linn. were estimated. Murashige and Skoog (MS) basal medium supplemented with α-naphthaleneacetic acid (NAA) 2.0 mg/l with benzyladenine (BA) 0.5 mg/l showed the highest amount of callus biomass (1.87 g/tube). The morphology of callus was significantly different according to the plant growth regulators and their concentrations used in the medium. The highest amount of total phenolic (21.70 mg gallic acid equivalent per gram (GAE/g)) and flavonoid (4.90 mg quercetin equivalent per gram (QE/g)) content and the maximum percentage (77.78 %) of radical scavenging activity were estimated in the extract of inflorescence. The synergistic effect of NAA (2.0 mg/l) and BA (0.5 mg/l) enhances the synthesis of total phenolic (9.20 mg GAE/g) and flavonoid (1.25 mg QE/g) content in the callus tissue. The callus produced by the same concentration shows 45.24 % of free radical scavenging activity. While comparing the various concentrations of NAA with 2,4-dichlorophenoxyacetic acid (2,4-D) for the production of callus biomass, total phenolic and flavonoid content and free radical scavenging activity, all the concentrations of NAA were found to be superior than those of 2,4-D.

Fractures in geriatric mice show decreased callus expansion and bone volume.

PubMed

Lopas, Luke A; Belkin, Nicole S; Mutyaba, Patricia L; Gray, Chancellor F; Hankenson, Kurt D; Ahn, Jaimo

2014-11-01

Poor fracture healing in geriatric populations is a significant source of morbidity, mortality, and cost to individuals and society; however, a fundamental biologic understanding of age-dependent healing remains elusive. The development of an aged-based fracture model system would allow for a mechanistic understanding that could guide future biologic treatments. Using a small animal model of long-bone fracture healing based on chronologic age, we asked how aging affected (1) the amount, density, and proportion of bone formed during healing; (2) the amount of cartilage produced and the progression to bone during healing; (3) the callus structure and timing of the fracture healing; and (4) the behavior of progenitor cells relative to the observed deficiencies of geriatric fracture healing. Transverse, traumatic tibial diaphyseal fractures were created in 5-month-old (n=104; young adult) and 25-month-old (n=107; which we defined as geriatric, and are approximately equivalent to 70-85 year-old humans) C57BL/6 mice. Fracture calluses were harvested at seven times from 0 to 40 days postfracture for micro-CT analysis (total volume, bone volume, bone volume fraction, connectivity density, structure model index, trabecular number, trabecular thickness, trabecular spacing, total mineral content, bone mineral content, tissue mineral density, bone mineral density, degree of anisotropy, and polar moment of inertia), histomorphometry (total callus area, cartilage area, percent of cartilage, hypertrophic cartilage area, percent of hypertrophic cartilage area, bone and osteoid area, percent of bone and osteoid area), and gene expression quantification (fold change). The geriatric mice produced a less robust healing response characterized by a pronounced decrease in callus amount (mean total volume at 20 days postfracture, 30.08±11.53 mm3 versus 43.19±18.39 mm3; p=0.009), density (mean bone mineral density at 20 days postfracture, 171.14±64.20 mg hydroxyapatite [HA]/cm3 versus 210.79±37.60 mg HA/cm3; p=0.016), and less total cartilage (mean cartilage area at 10 days postfracture, 101,279±46,755 square pixels versus 302,167±137,806 square pixels; p=0.013) and bone content (mean bone volume at 20 days postfracture, 11.68±3.18 mm3 versus 22.34±10.59 mm3; p<0.001) compared with the young adult mice. However, the amount of cartilage and bone relative to the total callus size was similar between the adult and geriatric mice (mean bone volume fraction at 25 days postfracture, 0.48±0.10 versus 0.50±0.13; p=0.793), and the relative expression of chondrogenic (mean fold change in SOX9 at 10 days postfracture, 135+25 versus 90±52; p=0.221) and osteogenic genes (mean fold change in osterix at 20 days postfracture, 22.2±5.3 versus 18.7±5.2; p=0.324) was similar. Analysis of mesenchymal cell proliferation in the geriatric mice relative to adult mice showed a decrease in proliferation (mean percent of undifferentiated mesenchymal cells staining proliferating cell nuclear antigen [PCNA] positive at 10 days postfracture, 25%±6.8% versus 42%±14.5%; p=0.047). Our findings suggest that the molecular program of fracture healing is intact in geriatric mice, as it is in geriatric humans, but callus expansion is reduced in magnitude. Our study showed altered healing capacity in a relevant animal model of geriatric fracture healing. The understanding that callus expansion and bone volume are decreased with aging can help guide the development of targeted therapeutics for these difficult to heal fractures.

Early and late fracture following extensive limb lengthening in patients with achondroplasia and hypochondroplasia.

PubMed

Kitoh, H; Mishima, K; Matsushita, M; Nishida, Y; Ishiguro, N

2014-09-01

Two types of fracture, early and late, have been reported following limb lengthening in patients with achondroplasia (ACH) and hypochondroplasia (HCH). We reviewed 25 patients with these conditions who underwent 72 segmental limb lengthening procedures involving the femur and/or tibia, between 2003 and 2011. Gender, age at surgery, lengthened segment, body mass index, the shape of the callus, the amount and percentage of lengthening and the healing index were evaluated to determine predictive factors for the occurrence of early (within three weeks after removal of the fixation pins) and late fracture (> three weeks after removal of the pins). The Mann‑Whitney U test and Pearson's chi-squared test for univariate analysis and stepwise regression model for multivariate analysis were used to identify the predictive factor for each fracture. Only one patient (two tibiae) was excluded from the analysis due to excessively slow formation of the regenerate, which required supplementary measures. A total of 24 patients with 70 limbs were included in the study. There were 11 early fractures in eight patients. The shape of the callus (lateral or central callus) was the only statistical variable related to the occurrence of early fracture in univariate and multivariate analyses. Late fracture was observed in six limbs and the mean time between removal of the fixation pins and fracture was 18.3 weeks (3.3 to 38.4). Lengthening of the tibia, larger healing index, and lateral or central callus were related to the occurrence of a late fracture in univariate analysis. A multivariate analysis demonstrated that the shape of the callus was the strongest predictor for late fracture (odds ratio: 19.3, 95% confidence interval: 2.91 to 128). Lateral or central callus had a significantly larger risk of fracture than fusiform, cylindrical, or concave callus. Radiological monitoring of the shape of the callus during distraction is important to prevent early and late fracture of lengthened limbs in patients with ACH or HCH. In patients with thin callus formation, some measures to stimulate bone formation should be considered as early as possible. ©2014 The British Editorial Society of Bone & Joint Surgery.

Callus Distraction Versus Single-Stage Lengthening With Bone Graft for Treatment of Brachymetatarsia: A Systematic Review.

PubMed

Jones, Marc D; Pinegar, David M; Rincker, Sarah A

2015-01-01

Brachymetatarsia deformity is a cosmetically displeasing anomaly that can become physically symptomatic. The surgical techniques most commonly used to repair the anomaly include single-stage lengthening with a bone graft, callus distraction, or a combination of bone grafting and callus distraction. A systematic review of the published data was performed to compare the outcomes of these 3 surgical procedures. A total of 61 studies reporting the use of callus distraction or single-stage lengthening, or both, for the treatment of brachymetatarsia were included in the present review. The incidence of major postoperative complications after callus distraction, single-stage lengthening, and the combination procedure was 49 (12.62%), 13 (3.72%), and 3 (33.33%), respectively. The number of minor complications with callus distraction, single-stage lengthening, and the combination procedure was 152 (39.18%), 55 (15.76%), and 1 (11.11%); the mean percentage of the original length achieved was 37.36%, 25.98% and 36.00%; and the mean length achieved was 17.5, 13.2, and 14.0 mm, respectively. The healing index (mo/cm) and healing time was 2.31 and 16.04 weeks, 1.90 and 9.35 weeks, and 3.93 and 14.62 weeks for callus distraction, single-stage lengthening, and the combination procedure, respectively. Our findings indicate that the callus distraction technique is associated with greater length gained but results in greater complication rates and requires almost twice the time to heal. Single-stage lengthening with a bone graft was associated with fewer complications and faster healing times than callus distraction but with lesser gains in length. From the information reported in the studies we reviewed, the prevalence of bilateral brachymetatarsia was 44.52%, and the female/male ratio was 13.7:1. Both of these findings seem to contradict the usual data given (72% for bilateral brachymetatarsia and a female/male ratio of 25:1). Copyright © 2015 American College of Foot and Ankle Surgeons. Published by Elsevier Inc. All rights reserved.

The evaluation of three treatments for plantar callus: a three-armed randomised, comparative trial using biophysical outcome measures.

PubMed

Hashmi, Farina; Nester, Christopher J; Wright, Ciaran R F; Lam, Sharon

2016-05-17

Callus is one of the most common foot skin complaints experienced by people of all ages. These painful and unsightly lesions often result in disability. The 'gold standard' of treatment is scalpel debridement by a trained specialist; however, people also seek over-the-counter remedies. There is a lack of clinical evidence for the efficacy of such products, which makes selection by patients and practitioners difficult. This randomised, three-armed, parallel, comparative trial aimed to test the efficacy of two home treatments for plantar callus using novel, objective outcome measures (skin hydration using the capacitance method; elasticity using negative pressure application; and surface texture using imaging). Additional outcome measures were: size of callus, quality of life (Foot Health Status Questionnaire) and self-reported participant satisfaction and compliance. The results were compared to a podiatry treatment. Participants were randomly allocated to one of three groups: potassium hydroxide (KOH, 40 %); trichloroacetic acid (TCA); and podiatry treatment. Participants were followed for 3 weeks after their initial intervention appointment (days 7, 14 and 21). The primary outcomes were the change from baseline in callus hydration, elasticity, texture, and size at each of the three time points. The secondary outcomes where: change in quality of life 21 days after treatment; resolution of calluses via visual inspection; and participant compliance and perception. Forty-six participants (61 ft) with plantar calluses were recruited. The podiatry treatment showed immediate and significant changes in all objective outcomes, associated foot pain and function (p <0.01). Lesser changes in skin quality and perceived pain and functional benefits occurred with TCA and KOH over 21 days. This is the first study where objective outcome measures have been used to measure changes in the nature of skin in response to callus treatments. We found significant differences in plantar callus in response to podiatry and two home treatments. The podiatry treatment showed immediate and significant changes in skin and associated foot pain and function. Lesser, but sometimes comparable, changes in skin and perceived pain and functional benefits occurred with TCA and KOH over 21 days. ISRCTN14751843 : date of registration: 30 April 2015.

Metabolism of [3H]Gibberellin A20 in Light- and Dark-grown Tobacco Callus Cultures 1

PubMed Central

Lance, Barbara; Durley, Richard C.; Reid, David M.; Thorpe, Trevor A.; Pharis, Richard P.

1976-01-01

The growth of tobacco callus in culture (previously shown to contain gibberellin [GA]-like substances), and its ability to metabolize [3H]-GA20 were examined. Growth rates, in the presence and absence of exogenously applied GA, were examined in light and dark conditions. Dark-grown callus grew at a much faster rate than light-grown and [3H]GA20 was metabolized much more rapidly in darkness than in light. [3H]GA1 was identified by combined gas-liquid chromatography/mass spectrometry as a major product of [3H]GA20, and was found to be a more potent promoter of tobacco callus growth than GA20. PMID:16659684

Impaired fracture healing with high non-union rates remains irreversible after traumatic brain injury in leptin-deficient mice

PubMed Central

Graef, F.; Seemann, R.; Garbe, A.; Schmidt-Bleek, K.; Schaser, K-D.; Keller, J.; Duda, G.; Tsitsilonis, S.

2017-01-01

Patients with traumatic brain injury (TBI) and long-bone fractures can show increased callus formation. This effect has already been reproduced in wild-type (wt) mice. However, the mechanisms remain poorly understood. Leptin is significantly increased following TBI, while its role in bone healing remains unclear. The aim of this study was to evaluate fracture healing in leptin-deficient ob/ob mice and to measure any possible impact of TBI on callus formation. 138 female, 12 weeks old, ob/ob mice were divided into four groups: Control, fracture, TBI and combined trauma. Osteotomies were stabilized with an external fixator; TBI was induced with Controlled Cortical Impact Injury. Callus bridging was weekly evaluated with in vivo micro-CT. Biomechanical testing was performed ex vivo. Micro-CT showed high non-union rates after three and four weeks in the fracture and combined trauma group. No differences were observed in callus volume, density and biomechanical properties at any time point. This study shows that bony bridging is impaired in the present leptin-deficient trauma model. Furthermore, the phenomenon of increased callus formation after TBI could not be reproduced in ob/ob mice, as in wt mice. Our findings suggest that the increased callus formation after TBI may be dependent on leptin signaling. PMID:28574414

Clinical photographic observation of plantar corns and callus associated with a nominal scale classification and inter- observer reliability study in a student population.

PubMed

Tollafield, David R

2017-01-01

The management of plantar corns and callus has a low cost-benefit with reduced prioritisation in healthcare. The distinction between types of keratin lesions that forms corns and callus has attracted limited interest. Observation is imperative to improving diagnostic predictions and a number of studies point to some confusion as to how best to achieve this. The use of photographic observation has been proposed to improve our understanding of intractable keratin lesions. Students from a podiatry school reviewed photographs where plantar keratin lesions were divided into four nominal groups; light callus (Grade 1), heavy defined callus (Grade 2), concentric keratin plugs (Grade 3) and callus with deeper density changes under the forefoot (Grade 4). A group of 'experts' assigned from qualified podiatrists validated the observer rated responses by the students. Cohen's weighted statistic (k) was used to measure inter-observer reliability. First year students (unskilled) performed less well when viewing photographs ( k  = 0.33) compared to third year students (semi-skilled, k  = 0.62). The experts performed better than students ( k  = 0.88) providing consistency with wound care models in other studies. Improved clinical annotation of clinical features, supported by classification of keratin- based lesions, combined with patient outcome tools, could improve the scientific rationale to prioritise patient care. Problems associated with photographic assessment involves trying to differentiate similar lesions without the benefit of direct palpation. Direct observation of callus with and without debridement requires further investigation alongside the model proposed in this paper.

Nano-copper-bearing stainless steel promotes fracture healing by accelerating the callus evolution process.

PubMed

Wang, Lei; Li, Guoyuan; Ren, Ling; Kong, Xiangdong; Wang, Yugang; Han, Xiuguo; Jiang, Wenbo; Dai, Kerong; Yang, Ke; Hao, Yongqiang

2017-01-01

Treatment for fractures requires internal fixation devices, which are mainly produced from stainless steel or titanium alloy without biological functions. Therefore, we developed a novel nano-copper-bearing stainless steel with nano-sized copper-precipitation (317L-Cu SS). Based on previous studies, this work explores the effect of 317L-Cu SS on fracture healing; that is, proliferation, osteogenic differentiation, osteogenesis-related gene expression, and lysyl oxidase activity of human bone mesenchymal stem cells were detected in vitro. Sprague-Dawley rats were used to build an animal fracture model, and fracture healing and callus evolution were investigated by radiology (X-ray and micro-CT), histology (H&E, Masson, and safranin O/fast green staining), and histomorphometry. Further, the Cu 2+ content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality. Our results revealed that 317L-Cu SS did not affect the proliferation of human bone mesenchymal stem cells, but promoted osteogenic differentiation and the expression of osteogenesis-related genes. In addition, 317L-Cu SS upregulated the lysyl oxidase activity. The X-ray and micro-CT results showed that the callus evolution efficiency and fracture healing speed were superior for 317L-Cu SS. Histological staining displayed large amounts of fibrous tissues at 3 weeks, and cartilage and new bone at 6 weeks. Further, histomorphometric analysis indicated that the callus possessed higher osteogenic efficiency at 6 weeks, and a high Cu 2+ content and increased Runx2 expression were observed in the callus for 317L-Cu SS. Besides, the mechanical strength of the fracture site was much better than that of the control group. Overall, we conclude that 317L-Cu SS possesses the ability to increase Cu 2+ content and promote osteogenesis in the callus, which could accelerate the callus evolution process and bone formation to provide faster and better fracture healing.

Callus induction of leaf explant Piper betle L. Var Nigra with combination of plant growth regulators indole-3-acetic acid (IAA), benzyl amino purin (BAP) and kinetin

NASA Astrophysics Data System (ADS)

Junairiah, Zuraidassanaaz, Nabilah Istighfari; Izdihar, Fairuz Nabil; Manuhara, Yosephine Sri Wulan

2017-09-01

The purpose of this research was to determine the combination of plant growth regulators IAA, BAP and kinetin towards callus induction and growth of leaf explants Piper betle L. VarNigra. Explants from leaf of Piper betle L. VarNigra was cultured on MS medium with 24 treatment combinations of plant growth regulators IAA and BAP and 24 treatment combinations of plant growth regulators IAA and kinetin with 0.0;0.5;1.0;1.5;2.0 mg/L concentration respectively, the observed variable were the length of time the formation of callus, callus morphology, fresh and dry weight of callus. The results of this research showed that the combination of growth regulators IAA with BAP and kinetin had effects on leaf growth of Piper betle L. VarNigra. During 8 weeks observation, it indicated that the combination of concentration IAA 0.5 mg/L and BAP 2.0 mg/L showed fastest callus formation at 8.5 days. Combination of concentration IAA 1.0 mg/L and BAP 1.5 mg/L showed the highest of fresh weight at 0.6596 grams, and the highest dry weight was obtained from the combination of concentration IAA 0.5 mg/L and BAP 0.5 mg/L at 0.0727 grams. Combination of concentration IAA 1.0 mg/L and kinetin 1.5 mg/L had the highest of fresh weight at 0.2972 grams and the highest dry weight at 0.1660 grams. Callus of Piper betle L. VarNigra had two textures, that were compact and friable, and also showed various kind of colors, like white, greenish white, yellowish white, tanned white, brown and black. Based on this research, that concentration IAA 1.0 mg/L and 1.5 mg/L kinetin was the best combination for induction of callus from leaf of Piper betle L. Var Nigra.

Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength.

PubMed

Morse, A; Yu, N Y C; Peacock, L; Mikulec, K; Kramer, I; Kneissel, M; McDonald, M M; Little, D G

2015-02-01

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost(-/-) mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14days), as well as at 28days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area. Normal endochondral ossification progressed in wild type and Sost(-/-) mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost(-/-) fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost(-/-) calluses over wild type with greater bone volume at day 10 (221%, p<0.01). The resultant Sost(-/-) united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p<0.05), and the strength of the fractured Sost(-/-) tibiae was greater than that that of wild type fractured tibiae. In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

Nano-copper-bearing stainless steel promotes fracture healing by accelerating the callus evolution process

PubMed Central

Kong, Xiangdong; Wang, Yugang; Han, Xiuguo; Jiang, Wenbo; Dai, Kerong; Yang, Ke; Hao, Yongqiang

2017-01-01

Treatment for fractures requires internal fixation devices, which are mainly produced from stainless steel or titanium alloy without biological functions. Therefore, we developed a novel nano-copper-bearing stainless steel with nano-sized copper-precipitation (317L-Cu SS). Based on previous studies, this work explores the effect of 317L-Cu SS on fracture healing; that is, proliferation, osteogenic differentiation, osteogenesis-related gene expression, and lysyl oxidase activity of human bone mesenchymal stem cells were detected in vitro. Sprague–Dawley rats were used to build an animal fracture model, and fracture healing and callus evolution were investigated by radiology (X-ray and micro-CT), histology (H&E, Masson, and safranin O/fast green staining), and histomorphometry. Further, the Cu2+ content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality. Our results revealed that 317L-Cu SS did not affect the proliferation of human bone mesenchymal stem cells, but promoted osteogenic differentiation and the expression of osteogenesis-related genes. In addition, 317L-Cu SS upregulated the lysyl oxidase activity. The X-ray and micro-CT results showed that the callus evolution efficiency and fracture healing speed were superior for 317L-Cu SS. Histological staining displayed large amounts of fibrous tissues at 3 weeks, and cartilage and new bone at 6 weeks. Further, histomorphometric analysis indicated that the callus possessed higher osteogenic efficiency at 6 weeks, and a high Cu2+ content and increased Runx2 expression were observed in the callus for 317L-Cu SS. Besides, the mechanical strength of the fracture site was much better than that of the control group. Overall, we conclude that 317L-Cu SS possesses the ability to increase Cu2+ content and promote osteogenesis in the callus, which could accelerate the callus evolution process and bone formation to provide faster and better fracture healing. PMID:29225463

A macrocyclic ellagitannin trimer, oenotherin T(1), from Oenothera species.

PubMed

Taniguchi, Shoko; Imayoshi, Yoko; Yabu-uchi, Ryoko; Ito, Hideyuki; Hatano, Tsutomu; Yoshida, Takashi

2002-01-01

Oenotherin T(1) was isolated from leaves of Oenothera tetraptera as a major ellagitannin. Its structure, that of a macrocyclic trimer with a new acyl group, an isodehydrovaloneoyl group, was established. This compound was also produced by callus tissues induced from O. laciniata leaves.

The Role of Ultrasound Imaging of Callus Formation in the Treatment of Long Bone Fractures in Children.

PubMed

Wawrzyk, Magdalena; Sokal, Jan; Andrzejewska, Ewa; Przewratil, Przemysław

2015-01-01

In the process of diagnosis and treatment of fractures, an X-ray study is typically performed. In modern medicine very important is the development of new diagnostic methods without adverse effects on the body. One of such techniques is ultrasound imaging. It has a high value in imaging most areas of the body, including the musculoskeletal system. Reports on the use of ultrasound in the evaluation of the callus are rare and this could be a method equivalent to or even better than standard radiographs. The aim of the study was to analyze the correlation of ultrasound with radiographs in imaging of callus formation after fractures of long bones in children and to analyze the correlation of vascular resistance index (RI) and the degree of vascularization of the callus with a subjective radiological assessment of the bone union quality. The prospective study was planned to qualify 50 children treated for long bones fractures of the arm, forearm, thigh and lower leg. Ultrasound diagnosis was carried out using a Philips iU22 camera equipped with a linear probe with 17-5-MHz resolution and MSK Superficial program. During ultrasound examination measurements of the callus were performed. Using the Power Doppler callus vascularity was visualized and vascular resistance index (RI) was measured. The same measurements were made within the corresponding area of the healthy limb. The results obtained by ultrasound were compared with radiograph measurements and with the subjective assessment of the callus quality. Preliminary results were developed on a group of 24 patients, where 28 fractured bones and 28 corresponding healthy bones were examined. Fifteen boys and 9 girls participated in the study. The average age at injury was, respectively, 11 and 9 years. In both groups fractures without displacement were the most frequent. A similar frequency was observed in fractures requiring reposition and subperiosteal fractures. In contrast, fractures with a slight displacement of the fragments, were 3 times more common in girls. Statistical analysis of the measurements of length and width of the callus demonstrated that the differences between results obtained in the ultrasound in comparison with X-rays were not statistically significant. Moreover, preliminary results showed a significantly higher degree of vascularization of the callus than of the healthy periosteum. Preliminary results indicate the high efficacy of ultrasound in the evaluation of callus formation after fractures of long bones in children and the possibility of its alternative use to X-ray examinations.

Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine.

PubMed

Shivani; Awasthi, Praveen; Sharma, Vikrant; Kaur, Navjot; Kaur, Navneet; Pandey, Pankaj; Tiwari, Siddharth

2017-01-01

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana.

Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine

PubMed Central

Shivani; Awasthi, Praveen; Sharma, Vikrant; Kaur, Navjot; Kaur, Navneet; Pandey, Pankaj

2017-01-01

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana. PMID:28797040

Extensin network formation in Vitis vinifera callus cells is an essential and causal event in rapid and H2O2-induced reduction in primary cell wall hydration

PubMed Central

2011-01-01

Background Extensin deposition is considered important for the correct assembly and biophysical properties of primary cell walls, with consequences to plant resistance to pathogens, tissue morphology, cell adhesion and extension growth. However, evidence for a direct and causal role for the extensin network formation in changes to cell wall properties has been lacking. Results Hydrogen peroxide treatment of grapevine (Vitis vinifera cv. Touriga) callus cell walls was seen to induce a marked reduction in their hydration and thickness. An analysis of matrix proteins demonstrated this occurs with the insolubilisation of an abundant protein, GvP1, which displays a primary structure and post-translational modifications typical of dicotyledon extensins. The hydration of callus cell walls free from saline-soluble proteins did not change in response to H2O2, but fully regained this capacity after addition of extensin-rich saline extracts. To assay the specific contribution of GvP1 cross-linking and other wall matrix proteins to the reduction in hydration, GvP1 levels in cell walls were manipulated in vitro by binding selected fractions of extracellular proteins and their effect on wall hydration during H2O2 incubation assayed. Conclusions This approach allowed us to conclude that a peroxidase-mediated formation of a covalently linked network of GvP1 is essential and causal in the reduction of grapevine callus wall hydration in response to H2O2. Importantly, this approach also indicated that extensin network effects on hydration was only partially irreversible and remained sensitive to changes in matrix charge. We discuss this mechanism and the importance of these changes to primary wall properties in the light of extensin distribution in dicotyledons. PMID:21672244

Secondary metabolites from Marchantia paleacea calluses and their allelopathic effects on Arabidopsis seed growth.

PubMed

Wang, Lei; Wang, Li-Ning; Zhao, Yu; Lou, Hong-Xiang; Cheng, Ai-Xia

2013-01-01

Rapid-growth Marchantia paleacea calluses were induced on MSK2 medium through surface sterilisation of the capsula. Ten known compounds including two steroids (1-2), six bibenzyls (3, 5-9), a flavonoid (10), and a terpenoid (4) were isolated from these calluses. The allelopathic effect of the six bibenzyls was assessed in Arabidopsis thaliana. Results revealed that bibenzyls could inhibit seedling growth in a dose-dependent manner.

Study on suitable for regeneration system of genetic transformation of kiwifruit

NASA Astrophysics Data System (ADS)

Yuan, Yun Xiang

2011-02-01

The stems of Actinidia Qinmei were taken as explants and induced callus formation after having gained the aseptic seedling in primary culture successfully, and then the calli were placed on different combinations regeneration medium. The results showed that the induction medium added 1 mg/L 6-BA and 0.1 mg/L NAA was beneficial to the callus induction in dark culture condition. The suitable regeneration medium was MS containing 2.0 mg/L 6BA, 0.1 mg/L NAA and 3% (W/V) sucrose, it could improve obviously the frequency of regenerated shoots. This method separated the callus induction from shoot regeneration and obtained more callus to optimize the regenerated medium, and also was advantageous to Kiwifruit genetic transformation.

Preliminary evaluation of a load-bearing BMP-2 carrier for segmental defect regeneration.

PubMed

Chu, Tien-Min G; Sargent, Peter; Warden, Stuart J; Turner, Charles H; Stewart, Rena L

2006-01-01

Large segmental defects in bones can result from tumor removal, massive trauma, congenital malformation, or non-union fractures. Such defects often are difficult to manage and require multiple-phase surgery to achieve adequate union and function. In this study, we propose a novel design of bone morphogenetic protein 2 (BMP-2) carrier for tissue engineering of segmental defect regeneration. The tube-shaped BMP-2 carrier was fabrication from a poly(propylene fumarate)/tricalcium phosphate (PPF/TCP) composite via casting technique developed in our laboratory. An in vitro evaluation showed that the compressive strength of the carrier decreased about 48% in 12 weeks while maintained a pH in the 6.8-7.4 range. In vivo study was conducted by implanting carriers loaded with 10 microg of BMP-2 in 5 mm rat femur gap model for 15 weeks. X-ray evidence of bridging was first found in the BMP group at 3 weeks. Bridging in all animals (N = 4) in the BMP group was found at 9 weeks. No x-ray evidence of bridging was found in the No BMP group (N = 3). pQCT analysis indicated that the bone mineral density of the callus in the BMP group has reached the level of native femur at 15 weeks after implantation, while the callus in the No BMP group has a bone mineral density at a lower level of 84% to the native femur. Histology analysis shows that a normal fatty bone marrow was restored and mineralized callus formed and bridged the segmental defect.

Improved tissue culture conditions for the emerging C4 model Panicum hallii.

PubMed

Grant, Joshua N; Burris, Jason N; Stewart, C Neal; Lenaghan, Scott C

2017-04-27

Panicum hallii Vasey (Hall's panicgrass) is a compact, perennial C 4 grass in the family Poaceae, which has potential to enable bioenergy research for switchgrass (Panicum virgatum L.). Unlike P. hallii, switchgrass has a large genome, allopolyploidy, self-incompatibility, a long life cycle, and large stature-all suboptimal traits for rapid genetics research. Herein we improved tissue culture methodologies for two inbred P. hallii populations: FIL2 and HAL2, to enable further development of P. hallii as a model C 4 plant. The optimal seed-derived callus induction medium was determined to be Murashige and Skoog (MS) medium supplemented with 40 mg L -1 L-cysteine, 300 mg L -1 L-proline, 3% sucrose, 1 g L -1 casein hydrolysate, 3 mg L -1 2,4-dichlorophenoxyacetic acid (2,4-D), and 45 μg L -1 6-benzylaminopurine (BAP), which resulted in callus induction of 51 ± 29% for FIL2 and 81 ± 19% for HAL2. The optimal inflorescence-derived callus induction was observed on MP medium (MS medium supplemented with 2 g L -1 L-proline, 3% maltose, 5 mg L -1 2,4-D, and 500 μg L -1 BAP), resulting in callus induction of 100 ± 0.0% for FIL2 and 84 ± 2.4% for HAL2. Shoot regeneration rates of 11.5 ± 0.8 shoots/gram for FIL2 and 11.3 ± 0.6 shoots/gram for HAL2 were achieved using seed-induced callus, whereas shoot regeneration rates of 26.2 ± 2.6 shoots/gram for FIL2 and 29.3 ± 3.6 shoots/gram for HAL2 were achieved from inflorescence-induced callus. Further, cell suspension cultures of P. hallii were established from seed-derived callus, providing faster generation of callus tissue compared with culture using solidified media (1.41-fold increase for FIL2 and 3.00-fold increase for HAL2). Aside from abbreviated tissue culture times from callus induction to plant regeneration for HAL2, we noted no apparent differences between FIL2 and HAL2 populations in tissue culture performance. For both populations, the cell suspension cultures outperformed tissue cultures on solidified media. Using the methods developed in this work, P. hallii callus was induced from seeds immediately after harvest in a shorter time and with higher frequencies than switchgrass. For clonal propagation, P. hallii callus was established from R1 inflorescences, similar to switchgrass, which further strengthens the potential of this plant as a C 4 model for genetic studies. The rapid cycling (seed-to-seed time) and ease of culture, further demonstrate the potential utility of P. hallii as a C 4 model plant.

Cell dedifferentiation, callus induction and somatic embryogenesis in Crataegus spp.

PubMed

Taimori, N; Kahrizi, D; Abdossi, V; Papzan, A H

2016-09-30

The present study describes the effects of light conditions, different kinds and concentrations of auxins [Naphthylacetic acid (NAA) and dichlorophenoxyacetic acid (2,4-D)] with cytokinin (Kin) in MS medium on callus induction and embryogenesis in Crataegus pseudoheterophylla, C. aronia and C.meyeri. At first leave explants sections were cultured on different combinations of plant growth regulators in dark and light for callus initiation and light conditions to evaluation the percentage and duration of survival, callus diameter, callus fresh weight and dry. Results of effects of plant growth regulators and light conditions on callus initiation revealed that highest percentage of callus initiation leaves in treatment (0.5 mg/l 2.4-D+0.5 mg/l KIN) for species C.pseudoheterophylla in dark conditions (100%). Dark conditions (100%) were more effective on callogenesis than light conditions (Photoperiodicity of 16-h and at light intensity of 40 µmol m-2 s-1). The callus induction of in vitro (64-100%) leaves was better than the ex vitro ones (0-100%). The combination of 2,4-D and Kin of in vitro leaves callogenesis has been indicated faster (one weeks) than the other combinations. The results also showed that the highest percentage (100%) and survival duration (6 months) was found in species C. pseudoheterophylla and C. meyeri in 0.1 mg/l 2,4.D + 0.5 mg/l KIN and 0.5 mg/l 2,4.D + 0.5 mg/l Kin. The minimum survival (0%) was absorbed in species C. aronia in 1 mg/l NAA. Maximum callus (10.63 and 10.00 mm respectively) was shown in 0.1 mg/l 2,4.D + 0.5 mg/l Kin and 0.5 mg/l 2,4.D + 0.5 mg/l Kin and was not significant differences after five week among species. The results showed that the highest fresh (1081.49 mg) and dry weight (506.88 and 506.98 mg respectively) was absorbed in species C. pseudoheterophylla in 0.1 mg/l 2,4.D + 0.5 mg/l Kin and 0.5 mg/l 2,4.D + 0.5 mg/l Kin. The embryogenesis was not occurred in any plant growth regulator combinations and species. The results of this study suggested that using 2,4-D with cytokinin (Kin) would be more beneficial for callogenesis.

Endogenous PTH deficiency impairs fracture healing and impedes the fracture-healing efficacy of exogenous PTH(1-34).

PubMed

Ren, Yongxin; Liu, Bo; Feng, Yuxu; Shu, Lei; Cao, Xiaojian; Karaplis, Andrew; Goltzman, David; Miao, Dengshun

2011-01-01

Although the capacity of exogenous PTH1-34 to enhance the rate of bone repair is well established in animal models, our understanding of the mechanism(s) whereby PTH induces an anabolic response during skeletal repair remains limited. Furthermore it is unknown whether endogenous PTH is required for fracture healing and how the absence of endogenous PTH would influence the fracture-healing capacity of exogenous PTH. Closed mid-diaphyseal femur fractures were created and stabilized with an intramedullary pin in 8-week-old wild-type and Pth null (Pth(-/-)) mice. Mice received daily injections of vehicle or of PTH1-34 (80 µg/kg) for 1-4 weeks post-fracture, and callus tissue properties were analyzed at 1, 2 and 4 weeks post-fracture. Cartilaginous callus areas were reduced at 1 week post-fracture, but were increased at 2 weeks post-fracture in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice respectively. The mineralized callus areas, bony callus areas, osteoblast number and activity, osteoclast number and surface in callus tissues were all reduced in vehicle-treated and PTH-treated Pth(-/-) mice compared to vehicle-treated and PTH-treated wild-type mice, but were increased in PTH-treated wild-type and Pth(-/-) mice compared to vehicle-treated wild-type and Pth(-/-) mice. Absence of endogenous PTH1-84 impedes bone fracture healing. Exogenous PTH1-34 can act in the absence of endogenous PTH but callus formation, including accelerated endochondral bone formation and callus remodeling as well as mechanical strength of the bone are greater when endogenous PTH is present. Results of this study suggest a complementary role for endogenous PTH1-84 and exogenous PTH1-34 in accelerating fracture healing.

Influence of fracture geometry on bone healing under locking plate fixations: A comparison between oblique and transverse tibial fractures.

PubMed

Miramini, Saeed; Zhang, Lihai; Richardson, Martin; Mendis, Priyan; Ebeling, Peter R

2016-10-01

Mechano-regulation plays a crucial role in bone healing and involves complex cellular events. In this study, we investigate the change of mechanical microenvironment of stem cells within early fracture callus as a result of the change of fracture obliquity, gap size and fixation configuration using mechanical testing in conjunction with computational modelling. The research outcomes show that angle of obliquity (θ) has significant effects on interfragmentary movement (IFM) which influences mechanical microenvironment of the callus cells. Axial IFM at near cortex of fracture decreases with θ, while shear IFM significantly increases with θ. While a large θ can increase shear IFM by four-fold compared to transverse fracture, it also result in the tension-stress effect at near cortex of fracture callus. In addition, mechanical stimuli for cell differentiation within the callus are found to be strongly negatively correlated to angle of obliquity and gap size. It is also shown that a relatively flexible fixation could enhance callus formation in presence of a large gap but could lead to excessive callus strain and interstitial fluid flow when a small transverse fracture gap is present. In conclusion, there appears to be an optimal fixation configuration for a given angle of obliquity and gap size. Copyright © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.

Regeneration of Stevia Plant Through Callus Culture

PubMed Central

Patel, R. M.; Shah, R. R.

2009-01-01

Stevia rebaudiana Bertoni that conventionally propagated by seed or by cuttings or clump division which has a limitation of quality and quantity seed material. In present study, callus culture technique was tried to achieve rapid plant multiplication for quality seed material. Callus induction and multiplication medium was standardized from nodal as well as leaf sagments. It is possible to maintain callus on Murashige and Skoog medium supplemented with 6-benzyl amino purine and naphthalene acetic acid. Maximum callus induction was obtained on Murashige and Skoog medium incorporated with 6-benzyl amino purine (2.0-3.0 mg/l) and naphthalene acetic acid (2.0 mg/l) treatments. However, Murashige and Skoog medium containing 2.0 mg/l 6-benzyl amino purine+2.0 mg/l naphthalene acetic acid was found to be the best for callus induction. Higher regeneration frequency was noticed with Murashige and Skoog medium supplemented with 2.0 mg/l 6-benzyl amino purine+0.2 mg/l naphthalene acetic acid. Regenerated plants were rooted better on ¼ Murashige and Skoog strength supplemented with 0.1 mg/l indole-3-butyric acid. The rooted plantlets were hardened successfully in tera care medium with 63 per cent survival rate. The developed protocol can be utilized for mass production of true to type planting material on large scale independent of season, i.e. external environmental conditions. PMID:20177455

Anthocyanin production in callus cultures of Cleome rosea: modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS.

PubMed

Simões, Claudia; Brasil Bizarri, Carlos Henrique; da Silva Cordeiro, Lívia; Carvalho de Castro, Tatiana; Machado Coutada, Leonardo César; Ribeiro da Silva, Antônio Jorge; Albarello, Norma; Mansur, Elisabeth

2009-10-01

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 microM 2,4-D. Reddish-pink regions were observed on callus surface after 6-7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH(4)(+)/NO(3)(-), 70 g L(-1) sucrose and supplementation with 0.90 microM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.

The combination effect of auxin and cytokinin on in vitro callus formation of Physalis angulata L. - A medicinal plant

NASA Astrophysics Data System (ADS)

Mastuti, Retno; Munawarti, Aminatun; Firdiana, Elok Rifqi

2017-11-01

Physalis angulata L. (Ciplukan) is one member of Solanaceae that has a potential as herbal medicine. This plant grows wild in the crop fields, forest edges, etc. However, ciplukan is increasingly difficult to find recently. In vitro callus is an alternative source to produce secondary metabolite production as well as to regenerate plants through indirect organogenesis. This study aims to identify the response of hypocotyl explants on in vitro callus formation induced by a combination of auxin and cytokinins. Two types of cytokinins, Kinetin and BAP (0.5 ppm) were combined with three types of auxin, i.e. 2.4-D, IBA and IAA, at three concentrations 0.5, 1.0 and 1.5 ppm. In all combinations of cytokinin and auxin, 50-100% of hypocotyl explants derived from in vitro seedling were able to produce callus either in a compact or watery friable texture. In MS medium supplemented with 2.4-D, callus FW (fresh weight) began to decline in the fourth week after culture. Callus FW that increased until 5 weeks of culture was obtained in medium IAA 0.5 + Kin 0.5, IBA 1.0 + Kin 0.5 and IBA 1 + BA 0.5. Almost all calli induced on a medium + Kinetin also produced roots. While medium + BAP was able to induce shoots regeneration.

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair.

PubMed

Scanlon, Vanessa; Soung, Do Yu; Adapala, Naga Suresh; Morgan, Elise; Hansen, Marc F; Drissi, Hicham; Sanjay, Archana

2015-01-01

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.

T and B cells participate in bone repair by infiltrating the fracture callus in a two-wave fashion.

PubMed

Könnecke, Ireen; Serra, Alessandro; El Khassawna, Thaqif; Schlundt, Claudia; Schell, Hanna; Hauser, Anja; Ellinghaus, Agnes; Volk, Hans-Dieter; Radbruch, Andreas; Duda, Georg N; Schmidt-Bleek, Katharina

2014-07-01

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing. Copyright © 2014 Elsevier Inc. All rights reserved.

[Study on Precursors for Synthesis of Anthraquinone Metabolites from Rheum tanguticum].

PubMed

Hasi, Qi-mei-ge; Lj, Hai-ling; Cheng, Yan; Menggen, Qi-qi-ge; Zhang, Yang

2015-01-01

To explore the potential precursors of the anthraquinone metabolites from Rheum tanguticum and preliminanly identify the synthesis pathway thereof. Sterile seedlings sprouted from the seeds of Rheum tanguticum were chosen as materials for inducing callus. The effects of different precursors and feeding duration on the callus of Rheum tanguticum and the anthraquinone yield in adult rheum were studied. The greatest improvement of anthraquinone yield was achieved by acetic acid, increasing 43. 9% for the callus and 45. 8% in the adult rheum; the second greatest improvement was achieved by malonic acid, increasing 15. 8% for the callus and only 3. 6% in the adult rheum. The yield of anthraquinone was not influenced significantly by benzoic acid and p-benzoquinone, and in contrast, was inhibited in some degree by shikimic acid and α-ketoglutaric acid. A suitable feeding duration was 36 h, which worked well for the effects of precursors. The precursor for synthesis of anthraquinone metabolites from Rheum tan- guticum is acetic acid, which improves the yields of callus and anthraquinone in adult rheum, concluding that the anthraquinone metabolites are synthesized via polyketone pathway.

Role of O-methyltransferase in the lignification of Douglas-fir cultured tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monroe, S.H.

1983-01-01

O-methyltransferase (OMT) is a key enzyme in the biosynthesis of lignin. This enzyme was isolated and characterized in an effort to understand why Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) callus tissue does not form appreciable amounts of lignin yet does form large amounts of the related flavonoids and tannins. It was shown that the OMT in the callus tissue is a cell wall associated, membrane-bound enzyme, in contrast to that of all reported plant species and to Douglas-fir seedlings, which have either a microsomal or soluble OMT. The effect this had on the OMT kinetic constants was studied. It was foundmore » that the callus OMT had much higher K/sub m/ constants for caffeic acid in both the membrane-bound and free forms compared with seedlings. The callus membrane-bound K/sub m/ for caffeic acid is 333 ..mu..M. The callus membrane-free K/sub m/ for caffeic acid is 250 ..mu..M. The seedling K/sub m/ for caffeic acid is 90 ..mu..M.« less

Electrostimulation of rat callus cells and human lymphocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aro, H.; Eerola, E.; Aho, A.J.

1984-01-01

Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The (/sup 3/H)thymidine incorporation of the callus cells and 5-(/sup 125/I)iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of (/sup 3/H)thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The (/sup 3/H)thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took upmore » more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.« less

Biomechanical consequences of callus development in Hoffmann, Wagner, Orthofix and Ilizarov external fixators.

PubMed

Juan, J A; Prat, J; Vera, P; Hoyos, J V; Sánchez-Lacuesta, J; Peris, J L; Dejoz, R; Alepuz, R

1992-09-01

A theoretical analysis by a finite elements model (FEM) of some external fixators (Hoffmann, Wagner, Orthofix and Ilizarov) was carried out. This study considered a logarithmic progress of callus elastic characteristics. A standard configuration of each fixator was defined where design and application characteristics were modified. A comparison among standard configurations and influence of every variation was made with regard to displacement and load transmission at the fracture site. An experimental evaluation of standard configurations was performed with a testing machine. After experimental validation of the theoretical model was achieved, an application of physiological loads which act on a fractured limb during normal gait was analysed. A minimal contribution from an external fixator to the total rigidity of the bone-callus-fixator system was assessed when a callus showing minimum elastic characteristics had just been established. Insufficient rigidity from the fixation devices to assure an adequate immobilization during the early stages of fracture healing was verified. However, regardless of the external fixator, callus development was the overriding element for the rigidity of the fixator-bone system.

The influence of auxins on the biosynthesis of isoprene derivatives in callus cultures of Vaccinium corymbosum var. bluecrop.

PubMed

Migas, Piotr; Luczkiewicz, Maria; Cisowski, Wojciech

2006-01-01

Callus cultures of Vaccinium corymbosum var. bluecrop were optimized for their isoprene derivatives production by supplementing Schenk-Hildebrandt (SH) medium with constant concentration of kinetin (2.32 microM) and two different amounts of selected auxins. Every auxin, except for IBA, used in 10-time higher concentration (2,4D, NAA, IAA, NOA) stimulated biosynthesis of beta-sitosterol and inhibited triterpene synthesis. Quantitative analysis of isoprene derivatives in callus biomass collected on the 25th day of the experiment proved that the analyzed callus of Vaccinium corymbosum var. bluecrop synthesized the highest amount of isoprene derivatives after subculturing on SH medium modified with 22.6 microM of 2,4D and 2.32 microM of kinetin.

Corns and calluses

MedlinePlus

... condition should prevent the calluses from returning. Wear gloves to protect your hands during activities that cause ... with changing to better-fitting shoes or wearing gloves. Call your provider if: You have diabetes and ...

Manganese toxicity to chlorophyll synthesis in tobacco callus. [Nicotiana tabacum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clairmont, K.B.; Hagar, W.G.; Davis, E.A.

1986-01-01

Tobacco (Nicotiana tabacum) pith explants were grown on manganese containing medium. At moderate concentration (10 millimolar), manganese selectivity inhibited chlorophyll synthesis, resulting initially in growth of white callus. Several weeks later the white callus turned brown due to the accumulation of a pigment identified as protoporphyrin IX by its elution profile using high performance liquid chromatography, by its absorption spectrum, and by its fluorescence properties. At a concentration of 100 millimolar manganese the pigment accumulated without growth of the explant.

1-Aminocyclopropane-1-carboxylic acid concentrations in shoot-forming and non-shoot-forming tobacco callus cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grady, K.L.; Bassham, J.A.

1982-09-01

Shoot-forming tobacco (Nicotiana tabacum var. Wisconsin 38) callus tissues contain significantly lower concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid compared to non-shoot-forming callus tissues. This difference is evident 1 day after subculture to shoot-forming or non-shoot-forming medium, and is maintained through the first week of growth. The lack of auxin in shoot-forming medium is the probable cause for this difference in ACC concentrations.

The Influence of Physical Forces on Progenitor Cell Migration, Proliferation and Differentiation in Fracture Repair

DTIC Science & Technology

2008-11-01

Figure 6) and the marrow spaces within the periosteal callus (Figure 7). This is true for all of the groups euthanized on day 48 except for the...the most consistent location for the MSC populations was in the medullary marrow and the marrow within the periosteal callus. Figure 7: GFP...positive cells in the periosteal callus (brown stained). The injected MSCs populated any area that consisted of marrow spaces. The areas in between the

Cloning and expression of 1-aminocyclopropane-1-carboxylate oxidase cDNA induced by thidiazuron during somatic embryogenesis of alfalfa (Medicago sativa).

PubMed

Feng, Bi-Hong; Wu, Bei; Zhang, Chun-Rong; Huang, Xia; Chen, Yun-Feng; Huang, Xue-Lin

2012-01-15

Embryogenic callus (EC) induced from petioles of alfalfa (Medicago sativa L. cv. Jinnan) on B5h medium turned green, compact and non-embryogenic when the kinetin (KN) in the medium was replaced partially or completely by thidiazuron (TDZ). The application of CoCl₂, which is an inhibitor of 1-aminocyclopropane-1-carboxylate oxidase (ACO), counteracted the effect of TDZ. Ethylene has been shown to be involved in the modulation of TDZ-induced morphogenesis responses. However, very little is known about the genes involved in ethylene formation during somatic embryogenesis (SE). To investigate whether ethylene mediated by ACO is involved in the effect of TDZ on inhibition of embryogenic competence of the alfalfa callus. In this study we cloned full-length ACO cDNA from the alfalfa callus, named MsACO, and observed changes in this gene expression during callus formation and induction of SE under treatment with TDZ or TDZ plus CoCl₂. RNA blot analysis showed that during the EC subcultural period, the expression level of MsACO in EC was significantly increased on the 2nd day, rose to the highest level on the 8th day and remained at this high level until the 21st day. However, the ACO expression in the TDZ (0.93 μM)-treated callus was higher than in the EC especially on the 8th day. Moreover the ACO expression level increased with increasing TDZ concentration during the subcultural/maintenance period of the callus. It is worth noting that comparing the treatment with TDZ alone, the treatment with 0.93 μM TDZ plus 50 μM CoCl₂ reduced both of the ACO gene expressions and ACO activity in the treated callus. These results indicate that the effect of TDZ could be counteracted by CoCl₂ either on the ACO gene expression level or ACO activity. Thus, a TDZ inhibitory effect on embryogenic competence of alfalfa callus could be mediated by ACO gene expression. Crown Copyright © 2011. Published by Elsevier GmbH. All rights reserved.

PTH 1-34 Ameliorates the Osteopenia and Delayed Healing of Stabilized Tibia Fracture in Mice with Achondroplasia Resulting from Gain-Of-Function Mutation of FGFR3

PubMed Central

Chen, Hangang; Sun, Xianding; Yin, Liangjun; Chen, Shuai; Zhu, Ying; Huang, Junlan; Jiang, Wanling; Chen, Bo; Zhang, Ruobin; Chen, Lin; Nie, Mao; Xie, Yangli; Deng, Zhongliang

2017-01-01

Bone fracture healing is processed through multiple stages including the cartilaginous callus formation and its transition to bony callus. FGFR3 negatively regulates chondrogenesis and enhances osteogenesis during skeleton development. We previously found in mice carrying gain-of-function mutation of FGFR3 that FGFR3 delays the healing of un-stabilized fracture that heals mainly through endochondral ossification. Since fracture is regularly treated in clinics with rigid fixation, and stabilized fracture is healed largely through intramembranous ossification, we asked whether FGFR3, a key regulator of osteogenesis, also affect the regeneration of stabilized fracture. We found that gain-of-function mutation of FGFR3 inhibits the initiation of chondrogenesis and the subsequent bone formation. We further studied whether PTH1-34 can improve the osteopenia and delayed healing of the stabilized tibia fracture in mice with achondroplasia. Fracture healing was evaluated by radiography, micro-CT, biomechanical tests, histology, and real-time polymerase chain reaction (RT-PCR) analysis. We found that PTH 1-34 can alleviate the decreased bone mass and compromised architecture in ACH mice. Histological analysis revealed that administration of PTH1-34 increased the size of both the total callus and cartilaginous callus at 14 days after the surgery in ACH mice. RT-PCR data suggested that systemic PTH1-34 accelerated the initiation of chondrogenesis and chondrocyte maturation (earlier and higher levels of expression of chondrogenesis related markers) and enhanced the osteogenic differentiation in the fracture callus in ACH mice. These results indicate that the PTH1-34 administration resulted in an enhanced callus formation during bone fracture healing in ACH mice, which is at least in part mediated by an increase of cartilaginous callus at early stage and the promotion of bone formation in bony callus. In summary, in this study we revealed that FGFR3 delays the regeneration of stabilized fracture by inhibiting both the chondrogenesis and osteogenesis, and PTH1-34 treatment can improve the dysregulated bone metabolism and delayed bone injury healing resulting from gain-of-function mutation of FGFR3. PMID:29104492

Biological activities of indoleacetylamino acids and their use as auxins in tissue culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hangarter, R.P.; Peterson, M.D.; Good, N.E.

1980-05-01

The auxin activities of a number of indoleacetylamino acid conjugates have been determined in three test systems: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-L-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetlyglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting growth of shoots andmore » roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-L-alanine and indoleacetylglycine. The other conjugates inhibit shoot formatin weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-L-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid.« less

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures.

PubMed

Maheshwari, Priti; Songara, Bharti; Kumar, Shailesh; Jain, Prachi; Srivastava, Kamini; Kumar, Anil

2007-08-01

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.

Amino Acid and Secondary Metabolite Production in Embryogenic and Non-Embryogenic Callus of Fingerroot Ginger (Boesenbergia rotunda).

PubMed

Ng, Theresa Lee Mei; Karim, Rezaul; Tan, Yew Seong; Teh, Huey Fang; Danial, Asma Dazni; Ho, Li Sim; Khalid, Norzulaani; Appleton, David Ross; Harikrishna, Jennifer Ann

2016-01-01

Interest in the medicinal properties of secondary metabolites of Boesenbergia rotunda (fingerroot ginger) has led to investigations into tissue culture of this plant. In this study, we profiled its primary and secondary metabolites, as well as hormones of embryogenic and non-embryogenic (dry and watery) callus and shoot base, Ultra Performance Liquid Chromatography-Mass Spectrometry together with histological characterization. Metabolite profiling showed relatively higher levels of glutamine, arginine and lysine in embryogenic callus than in dry and watery calli, while shoot base tissue showed an intermediate level of primary metabolites. For the five secondary metabolites analyzed (ie. panduratin, pinocembrin, pinostrobin, cardamonin and alpinetin), shoot base had the highest concentrations, followed by watery, dry and embryogenic calli. Furthermore, intracellular auxin levels were found to decrease from dry to watery calli, followed by shoot base and finally embryogenic calli. Our morphological observations showed the presence of fibrils on the cell surface of embryogenic callus while diphenylboric acid 2-aminoethylester staining indicated the presence of flavonoids in both dry and embryogenic calli. Periodic acid-Schiff staining showed that shoot base and dry and embryogenic calli contained starch reserves while none were found in watery callus. This study identified several primary metabolites that could be used as markers of embryogenic cells in B. rotunda, while secondary metabolite analysis indicated that biosynthesis pathways of these important metabolites may not be active in callus and embryogenic tissue.

Mechanical and structural properties of bone in non-critical and critical healing in rat.

PubMed

Hoerth, Rebecca M; Seidt, Britta M; Shah, Miheer; Schwarz, Carolin; Willie, Bettina M; Duda, Georg N; Fratzl, Peter; Wagermaier, Wolfgang

2014-09-01

A fracture in bone results in a dramatic change of mechanical loading conditions at the site of injury. Usually, bone injuries heal normally but with increasing fracture gaps, healing is retarded, eventually leading to non-unions. The clinical situation of these two processes with different outcomes is well described. However, the exact relation between the mechanical environment and characteristics of the tissues at all levels of structural hierarchy remains unclear. Here we studied the differences in material formation of non-critical (1mm) and critical (5mm gap) healing. We employed a rat osteotomy model to explore bone material structure depending upon the different mechanical conditions. In both cases, primary bone formation was followed by secondary bone deposition with mineral particle sizes changing from on average short and thick to long and thin particles. Bony bridging occurred at first in the endosteal callus and the nanostructure and microstructure developed towards cortical ordered material organization. In contrast, in critical healing, instead of bridging, a marrow cavity closure was formed endosteal, exhibiting tissue structure oriented along the curvature and a periosteal callus with less mature material structure. The two healing processes separated between 4 and 6 weeks post-osteotomy. The outcome of healing was determined by the varied geometrical conditions in critical and non-critical healing, inducing completely different mechanical situations. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

21 CFR 358.501 - Scope.

Code of Federal Regulations, 2011 CFR

2011-04-01

... MISCELLANEOUS EXTERNAL DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Corn and Callus Remover Drug Products § 358.501 Scope. (a) An over-the-counter corn and callus remover drug product in a form suitable for topical...

21 CFR 358.501 - Scope.

Code of Federal Regulations, 2010 CFR

2010-04-01

... MISCELLANEOUS EXTERNAL DRUG PRODUCTS FOR OVER-THE-COUNTER HUMAN USE Corn and Callus Remover Drug Products § 358.501 Scope. (a) An over-the-counter corn and callus remover drug product in a form suitable for topical...

Effect of AgNO3 and BAP on Root as a Novel Explant in Date Palm (Phoenix dactylifera cv. Medjool) Somatic Embryogenesis.

PubMed

Roshanfekrrad, Marjan; Zarghami, Reza; Hassani, Hassan; Zakizadeh, Hedayat; Salari, Ali

2017-01-01

Somatic embryogenesis techniques are used for cloning a wide range of varieties of date palms around the world. The aim of the present study was to develop an efficient method with the lowest cost and the greatest potential to obtain in vitro plantlets of date palm cv. Medjool. Also, produce embryogenic callus and somatic embryos without using 2,4-dichlorophenoxyacetic acid (2,4-D). In this study, produced plantlets through somatic embryogenesis were used in vitro roots as explant cultured on Murashige and Skoog (MS) media containing three level of Silver Nitrate (AgNO3) (0, 3 and 6 mg L-1) plus two level of 6-benzylaminopurine (BAP) (0 and 2 mg L-1) plus 0.1 mg L-1 1-naphthylacetic acid (NAA) for callus induction. After 12 weeks of culture, callus induction and after 16 weeks, production of embryogenic callus and embryos were occurred from root explants. According to the results, medium containing 2 mg L-1 BAP and 3 mg L-1 silver nitrate+0.1 mg L-1 NAA showed the highest amount of embryogenic callus fresh weight (1.38 g). This treatment also cause the highest number and length of embryos by production of 90.04 embryogenic callus with length of 11.18 mm. On the other hand, shoots were appeared from germinated embryos and white roots began to appear within 8 weeks. Medium contains 3 mg L-1 BAP and 0.1 mg L-1 NAA with average of 12.27 cm shoot length and 15.48 cm root length was the best. Control treatment had the lowest average shoot (3.71 cm) and root (5.03 cm) length. This study showed that certain concentration of silver nitrate and BAP has stimulating effect on growth of produced embryonic callus from root segments of Medjool cultivar of date palm.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yee, Cristal S.; Xie, LiQin; Hatsell, Sarah

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21 days post-fracture, and examined bone quality and callus outcomes at 21 days andmore » 42 days post-fracture (11 and 14 weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ + SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Lastly, our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone.« less

Development of highly regenerable callus lines and biolistic transformation of turf-type common bermudagrass [Cynodon dactylon (L.) Pers.].

PubMed

Li, L; Qu, R

2004-01-01

Common bermudagrass, Cynodon dactylon, is a widely used warm-season turf and forage species in the temperate and tropical regions of the world. Improvement of bermudagrass via biotechnology depends on improved tissue culture responses, especially in plant regeneration, and a successful scheme to introduce useful transgenes. When the concentration of 6-benzylaminopurine was adjusted in the culture medium, yellowish, compact calluses were observed from young inflorescence tissue culture of var. J1224. Nine long-term, highly regenerable callus lines (including a suspension-cultured line) were subsequently established, of which six were used for biolistic transformation. Five independent transgenic events, with four producing green plants, were obtained following hygromycin B selection from one callus line. Three transgenic events displayed resistance to the herbicide glufosinate, and one of these showed beta-glucuronidase activity since the co-transformation vector used in the experiments contained both the gusA and bar genes.

Temporal variation of applied inter fragmentary displacement at a bone fracture in harmony with maturation of the fracture callus.

PubMed

Gardner, T N; Evans, M; Simpson, H

1998-09-01

The amplitude of inter fragmentary displacement in long bone fractures greatly influences the pattern and speed of healing. Unfortunately, the amplitude of natural cyclical displacement arising from patient activity is random because of the inherent flexibility of fixation devices under natural loading. Although fixators may be designed to control the amplitude of this displacement, the amplitudes most beneficial to healing have not been determined. Furthermore, the appropriate amplitude must vary during healing as the reparative tissue (callus) progresses histologically and stiffens during maturation. In this study on an experimental fracture, the amplitude of applied cyclical displacement is varied during healing to correspond with the inverse of the callus stiffness versus time curve. In vivo mechanical stiffness tests on the callus indicate that the end point of the fixation period is achieved more rapidly than with a constant level of applied displacement.

Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli.

PubMed

Peredo, Elena L; Revilla, M Angeles; Arroyo-García, Rosa

2006-10-01

Organogenic calli induced from internodal segments were subcultured three times. Regenerated plants obtained from each subculture were analysed by molecular methods. No major genetic rearrangements were detected in the callus-derived plants since none of the amplified fragment-length polymorphism (AFLP) loci were found to be polymorphic. However, epigenetic changes due to a demethylation process were detected by methylation-sensitive amplified polymorphism (MSAP) technique. The results allowed inference of the possible relationship among the plants derived from different calli subcultures and the in vitro control. The plants recovered from the first and second callus subcultures clustered with the in vitro control pools in the phenogram while the regenerants from the third callus subculture showed the highest genetic distance with the controls. This is the first study reporting data about the genetic stability of callus-derived Humulus lupulus L. plants.

[Induction on callus culture and regeneration of Orostachyis fimbriatae].

PubMed

Su, Rui-jun; Zou, Li-juan; Wu, Qing-gui; Luo, Ming-hua

2014-01-01

To explore the effects of different hormonal combinations on induction, proliferation and differentiation of Orostachyis fimbriatae callus culture. Aseptic seedling leaves were used as explants,the different concentrations of IAA,NAA, 6-BA and KT on induction proliferation of callus were optimized by orthogonal test to explore the optimum medium for differentiation of callus by tissue culture techniques. The best medium for induction was MS + IAA 1.0 mg/L + NAA 0.5 mg/L + KT 1.0 mg/L, and the best hormonal combination for proliferation was MS + IAA 0.5 mg/L + 6-BA 0.5 mg/I. + KT 1.0 mg/L. The best medium for differentiation was MS + IAA 0.1 mg/L + KT 2.0 mg/L, and 1/2MS + IAA 0.2 mg/L was the optimum medium for rooting culture. The system of regeneration of Orostachyis fimbriatae is establishd by tissue culture techniques in this study.

In vitro production of gymnemic acid from Gymnema sylvestre (Retz) R. Br. ex roemer and schultes through callus culture under abiotic stress conditions.

PubMed

Ali Ahmed, Abdul Bakrudeen; Rao, Adhikarla Suryanarayana; Rao, Mandali Venkateswara

2009-01-01

Plant secondary metabolites have enormous potential for research and new drug development. Many secondary metabolites have a complex and unique structure and their production is often enhanced by biotic and abiotic stress conditions. Gymnemic acid (C(43)H(68)O(14)), a pentacyclic triterpenoid isolated from the leaves of Gymnema sylvestre, exhibits potent inhibitory effect on diabetes. The gymnemic acid content is determined by chromatographic methods: Camag HPTLC system equipped with a sample applicator Linomat IV and TLC scanner and integration software CAT 4.0. In HPLC C(18) (ODS) reverse phase column; water 486 UV detector; mobile phase, water/methanol (35:65, HPLC grade) + 0.1% acetic acid are used. Sample (20 microL) is applied with a flow rate of 1 mL/min and read at 230 nm with UV detector. The production of gymnemic acid is significantly higher in callus treated with 2,4-dichloro phenoxy acetic acid (2,4-D) and kinetin (KN). The blue light increases gymnemic acid accumulation upto 4.4-fold as compared with fluorescent light treatment and out of which 2.8 is found in leaves. Gymnemic acid is isolated from callus, grown under stress conditions followed by preparative TLC, simple and reproducible character based on HPTLC and high performance liquid chromatography.

In vivo biomechanical evaluation of a novel angle-stable interlocking nail design in a canine tibial fracture model.

PubMed

Déjardin, Loïc M; Cabassu, Julien B; Guillou, Reunan P; Villwock, Mark; Guiot, Laurent P; Haut, Roger C

2014-03-01

To compare clinical outcome and callus biomechanical properties of a novel angle stable interlocking nail (AS-ILN) and a 6 mm bolted standard ILN (ILN6b) in a canine tibial fracture model. Experimental in vivo study. Purpose-bred hounds (n = 11). A 5 mm mid-diaphyseal tibial ostectomy was stabilized with an AS-ILN (n = 6) or an ILN6b (n = 5). Orthopedic examinations and radiographs were performed every other week until clinical union (18 weeks). Paired tibiae were tested in torsion until failure. Callus torsional strength and toughness were statistically compared and failure mode described. Total and cortical callus volumes were computed and statistically compared from CT slices of the original ostectomy gap. Statistical significance was set at P < .05 RESULTS: From 4 to 8 weeks, lameness was less pronounced in AS-ILN than ILN6b dogs (P < .05). Clinical union was reached in all AS-ILN dogs by 10 weeks and in 3/5 ILN6b dogs at 18 weeks. Callus mechanical properties were significantly greater in AS-ILN than ILN6b specimens by 77% (failure torque) and 166% (toughness). Failure occurred by acute spiral (control and AS-ILN) or progressive transverse fractures (ILN6b). Cortical callus volume was 111% greater in AS-ILN than ILN6b specimens (P < .05). Earlier functional recovery, callus strength and remodeling suggest that the AS-ILN provides a postoperative biomechanical environment more conducive to bone healing than a comparable standard ILN. © Copyright 2014 by The American College of Veterinary Surgeons.

Formation of friable embryogenic callus in cassava is enhanced under conditions of reduced nitrate, potassium and phosphate

PubMed Central

Utsumi, Yoshinori; Utsumi, Chikako; Tanaka, Maho; Ha, Vu The; Matsui, Akihiro; Takahashi, Satoshi; Seki, Motoaki

2017-01-01

Agrobacterium-mediated transformation is an important research tool for the genetic improvement of cassava. The induction of friable embryogenic callus (FEC) is considered as a key step in cassava transformation. In the present study, the media composition was optimized for enhancing the FEC induction, and the effect of the optimized medium on gene expression was evaluated. In relative comparison to MS medium, results demonstrated that using a medium with reducing nutrition (a 10-fold less concentration of nitrogen, potassium, and phosphate), the increased amount of vitamin B1 (10 mg/L) and the use of picrolam led to reprogram non-FEC to FEC. Gene expression analyses revealed that FEC on modified media increased the expression of genes related to the regulation of polysaccharide biosynthesis and breakdown of cell wall components in comparison to FEC on normal CIM media, whereas the gene expression associated with energy flux was not dramatically altered. It is hypothesized that we reprogram non-FEC to FEC under low nitrogen, potassium and phosphate and high vitamin B1. These findings were more effective in inducing FEC formation than the previous protocol. It might contribute to development of an efficient transformation strategy in cassava. PMID:28806727

Extraction and quantification of gymnemic acids through gymnemagenin from callus cultures of Gymnema sylvestre.

PubMed

Kanetkar, P V; Singhal, R S; Laddha, K S; Kamat, M Y

2006-01-01

The phyto-constituents of Gymnema sylvestre are used in the treatment of diabetes and obesity. The present work reports on the extraction of gymnemic acid through gymnemagenin from callus cultures of G. sylvestre. Components were separated on pre-coated silica gel 60 GF254 plates with chloroform:methanol (8:2) and scanned using a densitometric scanner at 205 nm in the near-UV region. Linearity of determination of gymnemagenin was observed in the range 2-10 microg. The average percentage recovery of gymnemagenin from leaf callus extracts was 98.9+/-0.3.

Effects of Sucrose and Kinetin on Growth and Chlorophyll Synthesis in Tobacco Tissue Cultures 1

PubMed Central

Kaul, K.; Sabharwal, P. S.

1971-01-01

Investigations were carried out on the effects of various combinations of sucrose and kinetin concentrations on growth and chlorophyll production in a green and a nongreen clone of pith callus of Nicotiana tabacum L. It was found that 2 milligrams per liter or higher amounts of kinetin induced greening in the nongreen tissue. The observations suggested that growth of the callus and synthesis of chlorophyll and soluble protein are controlled by relative concentrations of sucrose and kinetin in the medium. Kinetin was found to be inhibitory for chlorophyll synthesis in the green callus. PMID:16657686

Knee arthrodesis after infected tumor mega prosthesis of the knee using an intramedullary nail for callus distraction.

PubMed

Kühne, C A; Taeger, G; Nast-Kolb, D; Ruchholtz, S

2003-03-01

Infected tumor endoprosthesis of the knee in young patients can prove to be challenging. Common procedures are débridement and prosthesis reimplantation, amputation, revision arthroplasty, and arthrodesis. We report the case of a 44-year-old man treated by arthrodesis followed by callus distraction after removal of an infected tumor mega prosthesis (Kotz type). Callus distraction was performed over a distance of 11 cm in 4 months using a femorotibial intramedullary nail with an external traction rope-winch system. The clinical, radiological, technical, and therapeutic features are discussed.

Genetic Transformation of Switchgrass

NASA Astrophysics Data System (ADS)

Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

Genetic transformation of switchgrass.

PubMed

Xi, Yajun; Ge, Yaxin; Wang, Zeng-Yu

2009-01-01

Switchgrass (Panicum virgatum L.) is a highly productive warm-season C4 species that is being developed into a dedicated biofuel crop. This chapter describes a protocol that allows the generation of transgenic switchgrass plants by Agrobacterium tumefaciens-mediated transformation. Embryogenic calluses induced from caryopses or inflorescences were used as explants for inoculation with A. tumefaciens strain EHA105. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Calluses resistant to hygromycin were obtained after 5-6 weeks of selection. Soil-grown switchgrass plants were regenerated about 6 months after callus induction and Agrobacterium-mediated transformation.

Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.).

PubMed

Liu, J R; Cantliffe, D J

1984-06-01

Leaf, shoot-tip, stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, bright- to pale-yellow, was compact and organized, while the other was dull-yellow and friable. The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids readily developed into a torpedo-shape before germination. The plantlets were transplanted to soil where they flowered and formed storage roots at maturity.

The effect of mesenchymal stem cell sheets on structural allograft healing of critical-sized femoral defects in mice

PubMed Central

Long, Teng; Zhu, Zhenan; Awad, Hani A.; Schwarz, Edward M.; Hilton, Matthew J.; Dong, Yufeng

2014-01-01

Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of x-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing. PMID:24393269

Whole-body vibration improves fracture healing and bone quality in rats with ovariectomy-induced osteoporosis.

PubMed

Butezloff, Mariana Maloste; Zamarioli, Ariane; Leoni, Graziela Bianchi; Sousa-Neto, Manoel Damião; Volpon, Jose Batista

2015-11-01

To investigate the effect of vibration therapy on the bone callus of fractured femurs and the bone quality of intact femurs in ovariectomized rats. Fifty-six rats aged seven weeks were divided into four groups: control with femoral fracture (CON, n=14), ovariectomized with femoral fracture (OVX, n=14), control with femoral fracture plus vibration therapy (CON+VT, n=14), and ovariectomized with femoral fracture plus vibration therapy (OVX+VT, n=14). Three months after ovariectomy or sham surgery, a complete fracture was produced at the femoral mid-diaphysis and stabilized with a 1-mm-diameter intramedullary Kirschner wire. X-rays confirmed the fracture alignment and fixation. Three days later, the VT groups underwent vibration therapy (1 mm, 60 Hz for 20 minutes, three times per week for 14 or 28 days). The bone and callus quality were assessed by densitometry, three-dimensional microstructure, and mechanical test. Ovariectomized rats exhibited a substantial loss of bone mass and severe impairment in bone microarchitecture, both in the non-fractured femur and the bone callus. Whole-body vibration therapy exerted an important role in ameliorating the bone and fracture callus parameters in the osteoporotic bone. Vibration therapy improved bone quality and the quality of the fracture bone callus in ovariectomized rats.

In Vitro Production of Echioidinin, 7-O-Methywogonin from Callus Cultures of Andrographis lineata and Their Cytotoxicity on Cancer Cells

PubMed Central

Mohammed, Arifullah; Chiruvella, Kishore K.; Rao, Yerra Koteswara; Geethangili, Madamanchi; Raghavan, Sathees C.; Ghanta, Rama Gopal

2015-01-01

Andrographis lineata is an herbal medicinal plant used in traditional medicine as a substitute for Andrographis paniculata. Here, using mature leaf explants of A. lineata we demonstrate for the first time the callus induction established on MS medium containing 1.0 mg l–1 IAA. Dried callus was subjected to solvent extraction with acetone. Further the acetone residue was separated by silica gel column chromatography, crystallized and characterized on the basis of nuclear magnetic resonance (proton and c13) and liquid chromatographic mass spectroscopy. This analysis revealed the occurrence of two known flavones namely, 7-O-methylwogonin (MW) and Echioidinin (ED). Furthermore, these compounds were tested for their cytotoxicity against leukemic cell line, CEM. We identify that ED and MW induced cytotoxicity in a time- and concentration-dependent manner. Further increase in the LDH release upon treatment with ED and MW further confirmed our cytotoxicity results against leukemic cell line. Strikingly, MW was more potent than ED when compared by trypan blue and MTT assays. Our results recapitulate the utility of callus cultures for the production of plant specific bioactive secondary metabolites instead of using wild plants. Together, our in vitro studies provide new insights of A. lineata callus cultures serving as a source for cancer chemotherapeutic agents. PMID:26488879

Genetic Transformation of Hordeum vulgare ssp. spontaneum for the Development of a Transposon-Based Insertional Mutagenesis System.

PubMed

Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder

2016-10-01

Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.

SUSPENSION CULTURE AND PLANT REGENERATION OF TYPHA LATIFOLIA

EPA Science Inventory

This study is the first reported attempt to generate a growth curve from Typha latifolia L. (broadleaf cattail) callus cells in suspension culture. Several media and hormone combinations were tested for their capacity to induce callus cell formation from T. latifolia leaf section...

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas).

PubMed

Mohd Din, Abd Rahman Jabir; Iliyas Ahmad, Fauziah; Wagiran, Alina; Abd Samad, Azman; Rahmat, Zaidah; Sarmidi, Mohamad Roji

2016-01-01

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L(-1)) and NAA (2 mg L(-1)) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5-1.5 mg L(-1)), BAP, NAA and 0.5 mg L(-1) of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L(-1)), Kin (1.5 mg L(-1)), NAA (0.5 mg L(-1)) and TDZ (0.5 mg L(-1)) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.

Melatonin-stimulated biosynthesis of anti-microbial ZnONPs by enhancing bio-reductive prospective in callus cultures of Catharanthus roseus var. Alba.

PubMed

Riaz, Hafiza Rida; Hashmi, Syed Salman; Khan, Tariq; Hano, Christophe; Giglioli-Guivarc'h, Nathalie; Abbasi, Bilal Haider

2018-05-18

Melatonin as plant growth regulator induces differential effects on metabolites that are responsible for reduction, capping and stabilization of zinc oxide nanoparticles. Phytochemical analysis of callus cultures was performed and results were compared with callus cultures supplemented with other plant growth regulators (α-napthalene acetic acid, 2,4-dichlorophenoxy acetic acid and thidiazuron). Highest total phenolic and flavonoid content [42.23 mg of gallic acid equivalent (GAE) g -1 DW and 36.4 mg of (quercetin equivalent) g -1 DW, respectively] were recorded at melatonin (1.0 µM) + NAA (13.5 µM). ZnONPs were synthesized from NAA (13.5 µM) and melatonin (1.0 µM) + NAA (13.5 µM)-induced calli extracts separately and characterized via X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). FTIR analysis confirmed the presence of phenolics and flavonoids that were mainly found responsible for reduction and capping of ZnONPs. SEM analysis showed triangular shaped ZnONPs synthesized from melatonin + NAA callus extract and these NPs were more dispersed as compared to the spherical-agglomerates of ZnONPs synthesized from NAA-mediated callus extract. Melatonin + NAA callus extract-mediated ZnONPs (having smaller size) were more potent against multiple drug resistant bacterial strains, e.g. Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by producing zone of inhibitions 17 ± 0.76 mm,10 ± 0.57 mm and 13 ± 0.54 mm, respectively.

Genetic diversity of improved salt tolerant calli of maize (Zea mays L.) using RAPD

NASA Astrophysics Data System (ADS)

Saputro, Triono Bagus; Dianawati, Siti; Sholihah, Nur Fadlillatus; Ermavitalini, Dini

2017-06-01

Maize is one of important cultivated plants in the world, in terms of production rates, utilization rates and demands. Unfortunately, the increment of demands were not followed by the increase of production rates since the cultivation area were significantly decrease. Coastal area is the marginal land that have a good potential to extend the cultivation area. The main challenge of this area is the high content of salt. The aims of this research were try to induce a new varian of local maize through in vitro culture and observe its genetic variation using RAPD. Bluto variety from Madura island was used as an explant in callus induction. Induction of callus were conducted using MS basal medium supplemented with 3 mg/L of 2,4 D under dark condition. While the selection stage was conducted using MS basal medium supplemented with 3 mg/L of 2,4 D with the addition of various concentration of NaCl (0 mg/L; 2500 mg/L; 5000 mg/L; and 7500 mg/L). The research were arranged in a completely randomized design with three replications. The exposion of NaCl were significantly decrease the mass of maize callus. The highest addition of callus weight was 210 mgs in control treatment, while the lowest is in 7500 mg/L with 3 mgs. The RAPD technique was utilized to characterize the genotype of maize callus. Out of five primers, only three primers can produce polymorphic bands named OPA10, OPB07 and OPC02. Taken together, the surviving callus of Bluto varians can be further developed as potential somaclone that has high tolerance to salt stress.

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

PubMed Central

Mohd Din, Abd Rahman Jabir; Iliyas Ahmad, Fauziah; Wagiran, Alina; Abd Samad, Azman; Rahmat, Zaidah; Sarmidi, Mohamad Roji

2015-01-01

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L−1) and NAA (2 mg L−1) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L−1), BAP, NAA and 0.5 mg L−1 of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L−1), Kin (1.5 mg L−1), NAA (0.5 mg L−1) and TDZ (0.5 mg L−1) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots. PMID:26858569

Biosynthesis of Gold and Silver Nanoparticles Using Extracts of Callus Cultures of Pumpkin (Cucurbita maxima).

PubMed

Iyer, R Indira; Panda, Tapobrata

2018-08-01

The potential of callus cultures and field-grown organs of pumpkin (Cucurbita maxima) for the biosynthesis of nanoparticles of the noble metals gold and silver has been investigated. Biosynthesis of AuNPs (gold nanoparticles) and AgNPs (silver nanoparticles) was obtained with flowers of C. maxima but not with pulp and seeds. With callus cultures established in MS-based medium the biogenesis of both AuNPs and AgNPs could be obtained. At 65 °C the biogenesis of AuNPs and AgNPs by callus extracts was enhanced. The AuNPs and AgNPs have been characterized by UV-visible spectroscopy, TEM, DLS and XRD. Well-dispersed nanoparticles, which exhibited a remarkable diversity in size and shape, could be visualized by TEM. Gold nanoparticles were found to be of various shapes, viz., rods, triangles, star-shaped particles, spheres, hexagons, bipyramids, discoid particles, nanotrapezoids, prisms, cuboids. Silver nanoparticles were also of diverse shapes, viz., discoid, spherical, elliptical, triangle-like, belt-like, rod-shaped forms and cuboids. EDX analysis indicated that the AuNPs and AgNPs had a high degree of purity. The surface charges of the generated AuNPs and AgNPs were highly negative as indicated by zeta potential measurements. The AuNPs and AgNPs exhibited remarkable stability in solution for more than four months. FTIR studies indicated that biomolecules in the callus extracts were associated with the biosynthesis and stabilisation of the nanoparticles. The synthesized AgNPs could catalyse degradation of methylene blue and exhibited anti-bacterial activity against E. coli DH5α. There is no earlier report of the biosynthesis of nanoparticles by this plant species. Callus cultures of Cucurbita maxima are effective alternative resources of biomass for synthesis of nanoparticles.

Divergent regeneration‐competent cells adopt a common mechanism for callus initiation in angiosperms

PubMed Central

Hu, Bo; Zhang, Guifang; Liu, Wu; Shi, Jianmin; Wang, Hua; Qi, Meifang; Li, Jiqin; Qin, Peng; Ruan, Ying; Huang, Hai; Zhang, Yijing

2017-01-01

Abstract In tissue culture, the formation of callus from detached explants is a key step in plant regeneration; however, the regenerative abilities in different species are variable. While nearly all parts of organs of the dicot Arabidopsis thaliana are ready for callus formation, mature regions of organs in monocot rice (Oryza sativa) and other cereals are extremely unresponsive to tissue culture. Whether there is a common molecular mechanism beyond these different regenerative phenomena is unclear. Here we show that the Arabidopsis and rice use different regeneration‐competent cells to initiate callus, whereas the cells all adopt WUSCHEL‐RELATED HOMEOBOX 11 (WOX11) and WOX5 during cell fate transition. Different from Arabidopsis which maintains regeneration‐competent cells in mature organs, rice exhausts those cells during organ maturation, resulting in regenerative inability in mature organs. Our study not only explains this old perplexity in agricultural biotechnology, but also provides common molecular markers for tissue culture of different angiosperm species. PMID:28975033

Metabolic changes associated with shoot formation in tobacco callus cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grady, K.L.

1982-08-01

Callus tissue derived from Nicotiana tabacum L. stem pith parenchyma cells was grown either on medium which maintains the callus in an undifferentiated state, or on medium which induces the formation of shoots. Two complementary types of studies were performed with the goal of establishing metabolic markers for the initiation of shoot formation: one designed to characterize the flow of radioactive sucrose into various metabolic pools, and one which allowed measurement of intermediary metabolite concentrations. In the former, callus tissue was incubated in (U-/sup 14/C)sucrose for periods up to one hour, and patterns of metabolite labelling in tissue grown onmore » shoot-forming and non-shoot-forming media were compared. In the latter studies, tissue was grown for an entire subculture period on non-shoot-forming medium labelled with (U-/sup 14/C)sucrose, then subcultured to labelled non-shoot-forming or shoot-forming media, and sampled at intervals during the first week of growth. 189 references.« less

Divergent regeneration-competent cells adopt a common mechanism for callus initiation in angiosperms.

PubMed

Hu, Bo; Zhang, Guifang; Liu, Wu; Shi, Jianmin; Wang, Hua; Qi, Meifang; Li, Jiqin; Qin, Peng; Ruan, Ying; Huang, Hai; Zhang, Yijing; Xu, Lin

2017-06-01

In tissue culture, the formation of callus from detached explants is a key step in plant regeneration; however, the regenerative abilities in different species are variable. While nearly all parts of organs of the dicot Arabidopsis thaliana are ready for callus formation, mature regions of organs in monocot rice ( Oryza sativa ) and other cereals are extremely unresponsive to tissue culture. Whether there is a common molecular mechanism beyond these different regenerative phenomena is unclear. Here we show that the Arabidopsis and rice use different regeneration-competent cells to initiate callus, whereas the cells all adopt WUSCHEL-RELATED HOMEOBOX 11 ( WOX11 ) and WOX5 during cell fate transition. Different from Arabidopsis which maintains regeneration-competent cells in mature organs, rice exhausts those cells during organ maturation, resulting in regenerative inability in mature organs. Our study not only explains this old perplexity in agricultural biotechnology, but also provides common molecular markers for tissue culture of different angiosperm species.

Acetylcholine suppresses shoot formation and callusing in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby.

PubMed

Bamel, Kiran; Gupta, Rajendra; Gupta, Shirish C

2016-06-02

We present experimental evidence to show that acetylcholine (ACh) causes decrease in shoot formation in leaf explants of tomato (Lycopersicon esculentum Miller var Pusa Ruby) when cultured on shoot regeneration medium. The optimum response was obtained at 10(-4) M ACh-enriched medium. ACh also causes decrease in percentage of cultures forming callus and reduces the callus mass. Inhibitors of enzymatic hydrolysis of ACh, neostigmine and physostigmine, also suppresses callogenesis and caulogenesis. On the other hand, the breakdown products of Ach, choline and acetate, do not alter the morphogenic response induced on the shoot regeneration medium. Neostigmine showed optimal reduction in shoot formation at 10(-5) M. The explants cultured on neostigmine augmented medium showed decline in the activity of ACh hydrolyzing enzyme acetylcholinesterase. ACh and neostigmine added together showed marked reduction in callus mass. These results strongly support the role of ACh as a natural regulator of morphogenesis in tomato plants.

Acetylcholine suppresses shoot formation and callusing in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby

PubMed Central

Bamel, Kiran; Gupta, Rajendra; Gupta, Shirish C.

2016-01-01

ABSTRACT We present experimental evidence to show that acetylcholine (ACh) causes decrease in shoot formation in leaf explants of tomato (Lycopersicon esculentum Miller var Pusa Ruby) when cultured on shoot regeneration medium. The optimum response was obtained at 10−4 M ACh-enriched medium. ACh also causes decrease in percentage of cultures forming callus and reduces the callus mass. Inhibitors of enzymatic hydrolysis of ACh, neostigmine and physostigmine, also suppresses callogenesis and caulogenesis. On the other hand, the breakdown products of Ach, choline and acetate, do not alter the morphogenic response induced on the shoot regeneration medium. Neostigmine showed optimal reduction in shoot formation at 10−5 M. The explants cultured on neostigmine augmented medium showed decline in the activity of ACh hydrolyzing enzyme acetylcholinesterase. ACh and neostigmine added together showed marked reduction in callus mass. These results strongly support the role of ACh as a natural regulator of morphogenesis in tomato plants. PMID:27348536

Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification or one-step freezing.

PubMed

Lambardi, Maurizio; De Carlo, Anna; Capuana, Maurizio

2005-01-01

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at -196 degrees C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45 degrees C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.

Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse model

DOE PAGES

Yee, Cristal S.; Xie, LiQin; Hatsell, Sarah; ...

2015-05-04

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21 days post-fracture, and examined bone quality and callus outcomes at 21 days andmore » 42 days post-fracture (11 and 14 weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ + SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Lastly, our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone.« less

Recombinant human parathyroid hormone (PTH 1-34) and low-intensity pulsed ultrasound have contrasting additive effects during fracture healing.

PubMed

Warden, Stuart J; Komatsu, David E; Rydberg, Johanna; Bond, Julie L; Hassett, Sean M

2009-03-01

Fracture healing is thought to be naturally optimized; however, recent evidence indicates that it may be manipulated to occur at a faster rate. This has implications for the duration of morbidity associated with bone injuries. Two interventions found to accelerate fracture healing processes are recombinant human parathyroid hormone [1-34] (PTH) and low-intensity pulsed ultrasound (LIPUS). This study aimed to investigate the individual and combined effects of PTH and LIPUS on fracture healing. Bilateral midshaft femur fractures were created in Sprague-Dawley rats, and the animals treated 7 days/week with PTH (10 microg/kg) or a vehicle solution. Each animal also had one fracture treated for 20 min/day with active-LIPUS (spatial-averaged, temporal-averaged intensity [I(SATA)]=100 mW/cm(2)) and the contralateral fracture treated with inactive-LIPUS (placebo). Femurs were harvested 35 days following injury to permit micro-computed tomography, mechanical property and histological assessments of the fracture calluses. There were no interactions between PTH and LIPUS indicating that their effects were additive rather than synergistic. These additive effects were contrasting with LIPUS primarily increasing total callus volume (TV) without influencing bone mineral content (BMC), and PTH having the opposite effect of increasing BMC without influencing TV. As a consequence of the effect of LIPUS on TV but not BMC, it decreased volumetric bone mineral density (vBMD) resulting in a less mature callus. The decreased maturity and persistence of cartilage at the fracture site when harvested offset any beneficial mechanical effects of the increased callus size with LIPUS. In contrast, the effect of PTH on callus BMC but not TV resulted in increased callus vBMD and a more mature callus. This resulted in PTH increasing fracture site mechanical strength and stiffness. These data suggest that PTH may have utility in the treatment of acute bone fractures, whereas LIPUS at an I(SATA) of 100 mW/cm(2) does not appear to be indicated in the management of closed, diaphyseal fractures.

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage

PubMed Central

2010-01-01

Background Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. Results For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation. Conclusion The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin. PMID:20444255

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage.

PubMed

Palama, Tony L; Menard, Patrice; Fock, Isabelle; Choi, Young H; Bourdon, Emmanuel; Govinden-Soulange, Joyce; Bahut, Muriel; Payet, Bertrand; Verpoorte, Robert; Kodja, Hippolyte

2010-05-05

Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l(-1)). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l(-1)) and TDZ (0.5 mg l(-1)). These calli were maintained by subculturing on BM containing IAA (0.5 mg l(-1)) and TDZ (0.3 mg l(-1)) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l(-1)). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation. The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid-protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm-like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.

Dihydroconiferyl alcohol - A cell division factor from Acer species.

PubMed

Lee, T S; Purse, J G; Pryce, R J; Horgan, R; Wareing, P F

1981-10-01

A compound that stimulated growth of soybean callus was isolated from spring sap of sycamore (Acer pseudoplatanus L.). Insufficient compound was isolated to permit it to be characterised. A compound with identical properties was isolated from commercial maple syrup, the concentrated spring sap of Acer saccharum L. The compound was identified as 3-(3-methoxy-4-hydroxyphenyl)-propan-1-ol (dihydroconiferyl alcohol, DCA). DCA was also active in the tobacco callus and radish leaf senescence assays, but was inactive in four other tests for cytokinin activity. DCA acted synergistically with kinetin to promote soybean callus growth. It is concluded that DCA has properties distinct from those of purine cytokinins.

In vitro production of M. × piperita not containing pulegone and menthofuran.

PubMed

Bertoli, Alessandra; Leonardi, Michele; Krzyzanowska, Justyna; Oleszek, Wieslaw; Pistelli, Luisa

2012-01-01

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.

Plant regeneration from hypocotyl- and anther-derived callus of berseem clover. [Trifolium alexandrium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mokhtarzedeh, A.; Constantin, M.J.

1978-01-01

Plants were regenerated from hypocotyl and anther explants of berseem clover (Trifolium alexandrinum L.) on Murashige and Skoog (MS) medium containing various combinations of plant growth regulators. The most efficient production of plants from hypocotyl explants involved: callus induction on MS medium with 1.0 mg/liter of naphthaleneacetic acid (NAA) and 1.5 mg/liter 6-furfurylaminopurine (KIN); callus increase on MS medium with 2.0 mg/liter of NAA and 0.1 mg/liter of N/sup 6/-(..delta../sup 2/-isopentenyl) adenine (2iP); induction of shoots on MS medium with 0.5 mg/liter each of NAA and KIN followed by induction of roots on MS medium with 1.0 mg/liter of indoleaceticmore » acid (IAA) and 0.1 mg/liter of 6-benzylaminopurine (BAP). Suspension cultures in liquid MS medium containing 2.0 mg/liter of NAA and 0.2 mg/liter of 2iP provided filterable cell preparations with 45% viable cells, 4% of which gave rise to colonies within 3 weeks after transfer to agar plates. Shoot development was observed when callus from the colonies was cultured on MS medium with 0.5 mg/liter of NAA and KIN. Preliminary results indicate that cells of root tips from hypocotyl- and anther-derived callus have the expected diploid and haploid number of chromosomes (2n = 16 and n = 8, respectively).« less

Radiological study of the effect of low calcium diet on the mineral metabolism of bone tissue. With reference to mineralization in callus (in Japanese)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakamura, K.

1972-01-01

Deficiency of available food material due either to poor diet or to malabsorption may adversely affect the skeleton. To study the affection, DDN mice were fed low calcium diet to induce low calcium state corresponding to malabsorption of calcium from the intestine. The femur was fractured manually. Then, calcium deposition in the callus was observed by microradiography and tracer technics with /sup 47/Ca. Increase of the body weight in mice fed low calcium diet was much slower than in the control. The affection of the low calcium diet on bone tissue appeared as a decrease of precipitation of calcium salt.more » This tendency was also observed in callus, Tracer study with /sup 47/Ca was performed in mice fed the low calcium diet for 24 days. Incorporation activity of calcium was generally high in each organ except the kidney. Callus in the site of the fracture in mice fed a low calcium diet was formed to the same degree as the control, although the amount of precipitated calcium in it was significantly poorer. In summary, insufficient mineralization in relation to osteogenesis occurred when the supply of the requisite electrolytes was insufficient or inappropriate. On the other hand, the uptake rate of calcinm in the callus was elevated even in the calcium deficient state. (auth)« less

Effect of x-ray irradiation on maize inbred line B73 tissue cultures and regenerated plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, A.S.; Cheng, D.S.K.; Milcic, J.B.

In order to enhance variation induced by the tissue culture process and to obtain agronomically desirable mutants, friable embryogenic tissue cultures of maize (Zea mays L.) inbred line B73 were x-ray irradiated with 11 doses (0-8.4 kilorads (kR)). Reductions in callus growth rate and embryogenic callus formation occurred with increasing x-ray doses 20 d and 3 months after irradiation. Callus irradiated with 0.8 kR showed a significant increase in growth rate and a 20% increase in embryogenic callus 9 months after irradiation. A total of 230 R/sub 0/ plants were regenerated for evaluation. Pollen fertility and seed set of R/submore » 0/ plants decreased with increasing x-ray dosage. Days to anthesis and plant height of R/sub 0/ plants varied among x-ray treatments but were generally reduced with higher dosages. The number of chromosomal aberrations increased with x-ray dosage. The R/sub 1/ seeds taken from R/sub 0/ plants were also grown and tested for mutant segregation. Plants regenerated from irradiated calli had a two- to 10-fold increase in mutations over plants regenerated from unirradiated control callus. Germination frequency of seeds from R/sub 0/ plants decreased with increasing x-ray dosage. Although chlorophyll mutants were most frequently observed, a number of vigorous plants with earlier anthesis date were also recovered.« less

High efficiency induction of callus and regeneration of sporophytes of Laminaria japonica (Phaeophyta)

NASA Astrophysics Data System (ADS)

Wang, Xi-Hua; Qin, Song; Li, Xin-Ping; Jiang, Peng; Zeng, Cheng-Kui; Qin, Mei

1998-03-01

Four media (PESI solid, MS liquid, MS solid and ASP-C-I solid medium) were used to induce callus from excised tissues of the kelp Laminaria japonica. Only PESI solid medium and MS solid medium produced calli. Modified MS solid medium supplemented with mannitol (3%,W/V), yeast extract (0.1%, W/V), VB2 (0.5 mg/ml), VB12 (0.5 mg/ml), kinetin (0.108 μg/ml) and NAA (1.860μg/ml) showed much better effect on callus induction than non-modified MS solid medium. After 24 days of induction 75.5% of tissues in PESI solid medium showed callus formation. For modified MS solid medium, after three months of induction 67.3% of tissues dedifferentiated into calli. No callus could be found after five months of induction in either MS liquid or ASP-C-I solid medium. When calli were squashed and cultured in N-P enriched autoclaved seawater, MS liquid medium and ASP12-NTA liquid medium (both modified with kelp extract), differentiation of cells and regeneration of sporophytes were only observed in ASP12-NTA medium supplemented with kelp extract. Gametophyte-like filaments formed first, then eggs were released. It was suggested that sporophyte formation could be a process of parthenogenesis. Sterilization techniques in tissue culture of L. japonica were also tested in this study.

Immunolocalization of Myostatin (GDF-8) Following Musculoskeletal Injury and the Effects of Exogenous Myostatin on Muscle and Bone Healing

PubMed Central

Elkasrawy, Moataz; Immel, David; Wen, Xuejun; Liu, Xiaoyan; Liang, Li-Fang

2012-01-01

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a “pool” of intense myostatin staining was observed among injured skeletal muscle fibers 12–24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 µg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury. PMID:22205678

The effect of various media and hormones via suspension culture on secondary metabolic activities of (Cape Jasmine) Gardenia jasminoides Ellis.

PubMed

Farzinebrahimi, Reza; Mat Taha, Rosna; Rashid, Kamaludin; Syafawati Yaacob, Jamilah

2014-01-01

The leaf of Gardenia jasminoides Ellis was used as explants and was cultured on MS and WPM media supplemented with various concentrations of NAA, IAA, 2,4-D, IBA, TDZ, and Kn (0 to 5 mg L(-1) with 0.5 increment). After six months, the higher percentage of callus (100%) and the best dry and fresh weight of callus were formed on WPM medium supplemented with 2,4-D and NAA (2.0-3.0 mg L(-1)) and this amount was decreased from (84%) to (69%) when this media supplemented with Kinetin and TDZ (1 mg L(-1)) respectively were used. Leaf segments cultured on WPM media added with Kn (1 mg L(-1)) and TDZ (2 mg L(-1)) yielded the least amount of callus. It was found that WPM media added with IAA (4.5-5.0 mg L(-1)) were optimum for root induction from G. jasminoides plantlets. Antibacterial screening of leaf extracts (in vivo) showed no inhibitory effect against E. coli, P. aeruginosa, S. aureus, and B. cereus, in contrast to callus extracts from leaf cultures supplemented with NAA, which showed inhibition activity against E. coli and B. cereus. The callus extracts from leaf cultures grown on both MS and WPM media showed higher antioxidant and superoxide dismutase activities than leaf extracts.

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

PubMed

Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

2009-03-01

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

Immunolocalization of myostatin (GDF-8) following musculoskeletal injury and the effects of exogenous myostatin on muscle and bone healing.

PubMed

Elkasrawy, Moataz; Immel, David; Wen, Xuejun; Liu, Xiaoyan; Liang, Li-Fang; Hamrick, Mark W

2012-01-01

The time course and cellular localization of myostatin expression following musculoskeletal injury are not well understood; therefore, the authors evaluated the temporal and spatial localization of myostatin during muscle and bone repair following deep penetrant injury in a mouse model. They then used hydrogel delivery of exogenous myostatin in the same injury model to determine the effects of myostatin exposure on muscle and bone healing. Results showed that a "pool" of intense myostatin staining was observed among injured skeletal muscle fibers 12-24 hr postsurgery and that myostatin was also expressed in the soft callus chondrocytes 4 days following osteotomy. Hydrogel delivery of 10 or 100 µg/ml recombinant myostatin decreased fracture callus cartilage area relative to total callus area in a dose-dependent manner by 41% and 80% (p<0.05), respectively, compared to vehicle treatment. Myostatin treatment also decreased fracture callus total bone volume by 30.6% and 38.8% (p<0.05), with the higher dose of recombinant myostatin yielding the greatest decrease in callus bone volume. Finally, exogenous myostatin treatment caused a significant dose-dependent increase in fibrous tissue formation in skeletal muscle. Together, these findings suggest that early pharmacological inhibition of myostatin is likely to improve the regenerative potential of both muscle and bone following deep penetrant musculoskeletal injury. © The Author(s) 2012

Reduction of rib fractures with a bioresorbable plating system: preliminary observations.

PubMed

Vu, Kim-Chi; Skourtis, Mia E; Gong, Xi; Zhou, Minhao; Ozaki, Wayne; Winn, Shelley R

2008-05-01

Operative fixation of rib fractures can reduce morbidity and mortality. Currently, resorbable fixation devices are used in a variety of surgical procedures. A standard osteotomy was prepared in 30 New Zealand white rabbits at the 12th rib. Eighteen had surgical repair with bioresorbable plates and 12 underwent nonoperative management. Half the animals in each group were killed at 3-week postfracture and the remaining animals were killed at 6-week postfracture. Ribs were radiographed and processed histologically to assess fracture healing. Rib reduction was defined as the alignment of the rib ends in a structural condition similar to the prefractured state and quantitative radiomorphometry measured the radiopaque callus surrounding the rib injury sites. Statistical analysis was performed using Fisher's exact test and an unpaired Student's t test and significance was established at p < 0.05. At both the 3- and 6-week intervals, seven of the nine rib fractures remained reduced in the operative group, whereas zero of six and three of six of the rib fractures remained reduced, respectively, in the nonoperative group. A statistically significant increase in radiopaque callus surrounding the rib injury sites was observed at 3 and 6 weeks in the fixed groups. Fixation of rib fractures with a bioresorbable miniplate system was superior to nonoperative treatment at the 3-week interval, with a statistically significant increase in radiopaque callus formation at both 3 and 6 weeks. Additional studies will evaluate the biomechanical outcomes and degradation tissue response after extended in vivo intervals.

Biotransformation of isofraxetin-6-O-β-d-glucopyranoside by Angelica sinensis (Oliv.) Diels callus.

PubMed

Zhou, Di; Zhang, Yuhua; Jiang, Zhe; Hou, Yue; Jiao, Kun; Yan, Chunyan; Li, Ning

2017-01-15

Isofraxetin-6-O-β-d-glucopyranoside, identified from traditional medicinal herbal Xanthoceras sorbifolia Bunge, has been demonstrated to be a natural neuroinflammatory inhibitor. In order to obtain more derivatives with potential anti-neuroinflammatory effects, biotransformation was carried out. According to the characteristics of coumarin skeleton, suspension cultures of Angelica sinensis (Oliv.) Diels callus (A. sinensis callus) were employed because of the presence of diverse phenylpropanoids biosynthetic enzymes. As a result, 15 products were yielded from the suspension cultures, including a new coumarin: 8'-dehydroxymethyl cleomiscosin A (1), together with 14 known compounds. Their structures were elucidated by extensive spectroscopic analysis. Furthermore, the biotransformed pathways were discussed. Among them, compound 13 was transformed from isofraxetin-6-O-β-d-glucopyranoside, while compounds 1-6, 10-12, 14-15 were derived from the culture medium stimulated by the substrate. The biotransformation processes include hydroxylation, oxidation and esterification. Furthermore, their inhibitory effects on lipopolysaccharide (LPS)-activated nitric oxide (NO) production were evaluated in BV2 microglial cells. It is worth noting that, 1, 1'-methanediylbis(4-methoxybenzene) (3), obtucarbamates A (5), 2-nonyl-4-hydroxyquinoline N-oxide (10) and 1H-indole-3-carbaldehyde (11) exhibited significant inhibitory effect against neuroinflammation with IC 50 values at 1.22, 10.57, 1.02 and 0.76μM respectively, much stronger than that of the positive control minocycline (IC 50 35.82μM). Copyright © 2016 Elsevier Ltd. All rights reserved.

EFFECT OF PHOSPHORUS CONCENTRATION ON THE GROWTH OF CATTAIL CALLUS CELLS

EPA Science Inventory

This investigation examined the growth of Typha latifolia (cattail) callus cells grown in 5 different (0, 11, 22, 33, 44, jg/L(-1) phosphosur concentrations. The cells were grown for two successive subcultures on semi-solid media, and subsequently in suspension culture with the s...

THE FORMATION OF BONE CALLUS DURING RADIATION SICKNESS (in Russian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nazarishvill, G.P.; Vepkhvadze, R.Ya.; Vakhtangashvili, T.A.

1957-01-01

>Six dogs were irradiated with an x-ray dose of 408 r, and the skin bone was thee broken immediately after radiation. The healing process was followed by x rays in the control group of dogs in which a well-developed bone callus could be observed on the 50th day. No sign of the formation of a bone callus at the fracture site could be observed in any of the irradiated dogs. Even on the l02nd day after the fracture a wide slit could be seen on thee x-ray diagram at the point of fracture where the bones had not knit togethermore » in the irradiated dogs. (TTT)« less

Synthesis of deuterium-labelled substrates for the study of oleuropein biosynthesis in Olea europaea callus cultures.

PubMed

Serrilli, Anna Maria; Maggi, Agnese; Casagrande, Valentina; Bianco, Armandodoriano

2016-01-01

We propose the cell culture approach to investigate oleuropein (1) biogenesis in Olea europaea L. We suggest employing olive callus cultures to identify the iridoidic precursor of oleuropein. In fact, we confirmed that callus cells from olive shoot explants are able to produce key secoiridoid as 1. To enable this approach, we synthesised and characterised deuterium-labelled iridoidic precursors belonging both to the loganin and the 8-epiloganin series. These iridoids are [7,8-(2)H2]-7-deoxy-8-epi-loganin (2(D)), [8,10-(2)H2]-8-epi-loganin (4(D)) and [7,8-(2)H2]-7-deoxy-loganin (3(D)).

The effect of mesenchymal stem cell sheets on structural allograft healing of critical sized femoral defects in mice.

PubMed

Long, Teng; Zhu, Zhenan; Awad, Hani A; Schwarz, Edward M; Hilton, Matthew J; Dong, Yufeng

2014-03-01

Structural bone allografts are widely used in the clinic to treat critical sized bone defects, despite lacking the osteoinductive characteristics of live autografts. To address this, we generated revitalized structural allografts wrapped with mesenchymal stem/progenitor cell (MSC) sheets, which were produced by expanding primary syngenic bone marrow derived cells on temperature-responsive plates, as a tissue-engineered periosteum. In vitro assays demonstrated maintenance of the MSC phenotype in the sheets, suggesting that short-term culturing of MSC sheets is not detrimental. To test their efficacy in vivo, allografts wrapped with MSC sheets were transplanted into 4-mm murine femoral defects and compared to allografts with direct seeding of MSCs and allografts without cells. Evaluations consisted of X-ray plain radiography, 3D microCT, histology, and biomechanical testing at 4- and 6-weeks post-surgery. Our findings demonstrate that MSC sheets induce prolonged cartilage formation at the graft-host junction and enhanced bone callus formation, as well as graft-host osteointegration. Moreover, a large periosteal callus was observed spanning the allografts with MSC sheets, which partially mimics live autograft healing. Finally, biomechanical testing showed a significant increase in the structural and functional properties of MSC sheet grafted femurs. Taken together, MSC sheets exhibit enhanced osteogenicity during critical sized bone defect repair, demonstrating the feasibility of this tissue engineering solution for massive allograft healing. Copyright © 2013 Elsevier Ltd. All rights reserved.

Inhibition of chlorophyll synthesis and carotenoid accumulation by manganese and cobalt

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clairmont, K.B.; Davis, E.; Hagar, W.

1986-05-01

The authors have developed methods for the separation and identification of the major pigments of the photosynthetic apparatus in plants using reversed phase microbore high performance liquid chromatography. Using these methods they have monitored the concentrations of pigments in tissue cultured tobacco callus in the absence and presence of excess manganese and cobalt. Manganese and cobalt were reported to inhibit chlorophyll synthesis in blue green algae. They have found that excess manganese blocks chlorophyll synthesis in tobacco callus also. In the manganese inhibited callus there is an increase in the concentration of protoporphyrin IX- the last common precursor to bothmore » the chlorophyll and heme synthetic pathways. They have found that cobalt also blocks chlorophyll synthesis in tissue cultured tobacco callus, but at a much lower concentration. In addition to the inhibition of chlorophyll synthesis by excess manganese and cobalt, the accumulation of carotenoids is reduced by several orders of magnitude in this tissue. The absence of chlorophyll may prevent assembly of any components of the photosynthetic apparatus in these cells.« less

Regeneration of roots from callus reveals stability of the developmental program for determinate root growth in Sonoran Desert Cactaceae.

PubMed

Shishkova, Svetlana; García-Mendoza, Edith; Castillo-Díaz, Vicente; Moreno, Norma E; Arellano, Jesús; Dubrovsky, Joseph G

2007-05-01

In some Sonoran Desert Cactaceae the primary root has a determinate root growth: the cells of the root apical meristem undergo only a few cell division cycles and then differentiate. The determinate growth of primary roots in Cactaceae was found in plants cultivated under various growth conditions, and could not be reverted by any treatment tested. The mechanisms involved in root meristem maintenance and determinate root growth in plants remain poorly understood. In this study, we have shown that roots regenerated from the callus of two Cactaceae species, Stenocereus gummosus and Ferocactus peninsulae, have a determinate growth pattern, similar to that of the primary root. To demonstrate this, a protocol for root regeneration from callus was established. The determinate growth pattern of roots regenerated from callus suggests that the program of root development is very stable in these species. These findings will permit future analysis of the role of certain Cactaceae genes in the determinate pattern of root growth via the regeneration of transgenic roots from transformed calli.

Vancomycin-bearing synthetic bone graft delivers rhBMP-2 and promotes healing of critical rat femoral segmental defects.

PubMed

Skelly, Jordan D; Lange, Jeffrey; Filion, Tera M; Li, Xinning; Ayers, David C; Song, Jie

2014-12-01

Bone grafts simultaneously delivering therapeutic proteins and antibiotics may be valuable in orthopaedic trauma care. Previously, we developed a poly(2-hydroxyethyl methacrylate)-nanocrystalline hydroxyapatite (pHEMA-nHA) synthetic bone graft that, when preabsorbed with 400-ng rhBMP-2/7, facilitated the functional repair of critical-size rat femoral defects. Recently, we showed that pHEMA-nHA effectively retains/releases vancomycin and rhBMP-2 in vitro. The success of such a strategy requires that the incorporation of vancomycin does not compromise the structural integrity of the graft nor its ability to promote bone healing. (1) To evaluate the ability of pHEMA-nHA-vancomycin composites in combination with 3-µg rhBMP-2 to repair 5 mm rat femoral segmental defects, and (2) To determine if the encapsulated vancomycin impairs the graft/rhBMP-2-assisted bone repair. pHEMA-nHA-vancomycin, pHEMA-nHA, or collagen sponge control with/without 3-µg rhBMP-2 were press-fit in 5 mm femoral defects in SASCO-SD male rats (289-300 g). Histology, microcomputed tomography, and torsion testing were performed on 8- and 12-week explants to evaluate the extent and quality of repair. The effect of vancomycin on the temporal absorption of endogenous BMP-2 and stromal cell-derived factor-1 was evaluated by immunohistochemistry. These factors are important for bone healing initiation and stem cell recruitment, respectively. Partial bridging of the defect with bony callus by 12 weeks was observed with pHEMA-nHA-vancomycin without rhBMP-2 while full bridging with substantially mineralized callus and partial restoration of torsional strength was achieved with 3-µg rhBMP-2. The presence of vancomycin changed the absorption patterns of endogenous proteins on the grafts, but did not appear to substantially compromise graft healing. The composite pHEMA-nHA-vancomycin preabsorbed with 3-µg rhBMP-2 promoted repair of 5 mm rat femoral segmental defects. With the sample sizes applied, vancomycin encapsulation did not appear to have a negative effect on bone healing. pHEMA-nHA-vancomycin preabsorbed with rhBMP-2 may be useful in the repair of critical-size long bone defects prone to infections.

Correlation between RUST assessments of fracture healing to structural and biomechanical properties.

PubMed

Cooke, Margaret E; Hussein, Amira I; Lybrand, Kyle E; Wulff, Alexander; Simmons, Erin; Choi, Jeffrey H; Litrenta, Jody; Ricci, William M; Nascone, Jason W; O'Toole, Robert V; Morgan, Elise F; Gerstenfeld, Louis C; Tornetta, Paul

2018-03-01

Radiographic Union Score for Tibia (RUST) and modified RUST (mRUST) are radiographic tools for quantitatively evaluating fracture healing using a cortical scoring system. This tool has high intra-class correlation coefficients (ICCs); however, little evidence has evaluated the scores against the physical properties of bone healing. Closed, stabilized fractures were made in the femora of C3H/HeJ male mice (8-12 week-old) of two dietary groups: A control and a phosphate restricted diet group. Micro-computed tomography (µCT) and torsion testing were carried out at post-operative days (POD) 14, 21, 35, and 42 (n = 10-16) per group time-point. Anteroposterior and lateral radiographic views were constructed from the µCT scans and scored by five raters. The raters also indicated if the fracture were healed. ICCs were 0.71 (mRUST) and 0.63 (RUST). Both RUST scores were positively correlated with callus bone mineral density (BMD) (r = 0.85 and 0.80, p < 0.001) and bone volume fraction (BV/TV) (r = 0.86 and 0.80, p < 0.001). Both RUST scores positively correlated with callus strength (r = 0.35 and 0.26, p < 0.012) and rigidity (r = 0.50 and 0.39, p < 0.001). Radiographically healed calluses had a mRUST ≥13 and a RUST ≥10 and had excellent relationship to structural and biomechanical metrics. Effect of delayed healing due to phosphate dietary restrictions was found at later time points with all mechanical properties (p < 0.011), however no differences found in the RUST scores (p > 0.318). Clinical relevance of this study is both RUST scores showed high correlation to physical properties of healing and generally distinguished healed vs. non-healed fractures. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:945-953, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Induction of bulb organogenesis in in vitro cultures of tarda tulip (Tulipa tarda Stapf.) from seed-derived explants.

PubMed

Maślanka, Małgorzata; Bach, Anna

2014-01-01

A protocol for obtaining bulbs via in vitro organogenesis was developed for tarda tulip ( Tulipa tarda Stapf). Scale explants were obtained from bulbs formed at the base of seedlings or from adventitious bulbs that developed from callus tissue forming on stolons or on germinating seeds. Some explants were subjected to chilling at 5°C for 12 wk. The culture media contained 3 or 6% sucrose and was supplemented with either no growth regulators, either 0.5 μM 6-benzyl-aminopurine (BAP) or 18.9 or 94.6 μM abscisic acid (ABA). Cultures were maintained in the dark at 20°C. Callus tissue developed mainly on media without growth regulators or with BAP. Callus was formed from up to 96% of explants derived from non-chilled adventitious bulbs that were treated with 3% sucrose and 0.5 μM BAP. Less callus was formed from chilled explants compared with non-chilled explants. Newly formed adventitious bulbs appeared on the explants via direct and indirect organogenesis. The media with BAP promoted the formation of adventitious bulbs at a rate of 56-92% from non-chilled explants, whereas a maximum rate of 36% was observed from chilled explants. ABA inhibited the induction of adventitious bulbs and callus. The adventitious bulbs obtained in these experiments contained a meristem, which was evidence that they had developed properly.

C/sub 4/ photosynthesis in Euphorbia degeneri and E. remyi: a comparison of photosynthetic carbon metabolism in leaves, callus cultures and regenerated plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruzin, S.E.

1984-04-01

Based on analysis of /sup 14/CO/sub 2/ fixation kinetics and assays of enzymes related to C/sub 4/ metabolism (NAD-ME, NADP-ME, NAD-MDH, NADP-MDH, AST, ALT), leaves and regenerated plants of Euphorbia degeneri exhibit a modified NADP-ME-type photosynthesis. Apparently, both aspartate and malate are used for transport of CO/sub 2/ to bundle sheath cells. Callus grown on either non-shoot-forming or shoot-forming media fixes CO/sub 2/ into RPP-cycle intermediates and sucrose, as well as malate and aspartate. /sup 14/CO/sub 2/ pulse/chase kinetics show no significant loss of label from C/sub 4/ acids throughout a one minute chase. Analysis of PEPCase revealed the presencemore » of 2 isoenzymes in both leaf and regenerated plant tissues (K/sub m/ (PEP) = 0.080 and 0.550) but only one isoenzyme in callus (K/sub m/ = 0.100). It appears that C/sub 4/ photosynthesis does not occur in callus derived from this C/sub 4/ dicot but is regenerated concomitant with shoot regeneration, and ..beta..-carboxylation of PEP in callus, mediated by the low K/sub m/ isoenzyme of PEPCase, produces C/sub 4/ acids that are not involved in the CO/sub 2/ shuttle mechanism characteristic of C/sub 4/ photosynthesis. 161 references, 19 figures, 12 tables.« less

ICS classification system of infected osteosynthesis: Long-term results.

PubMed

Romanò, Carlo L; Morelli, Ilaria; Romanò, Delia; Meani, Enzo; Drago, Lorenzo

2018-03-01

The best treatment strategy for infected osteosyntheses is still debated. While hardware removal or eventually early device exchange may be necessary in most of the cases, temporary hardware retention until fracture healing can be a valid alternative option in others. Aim of the present study is to report the long-term results of 215 patients with infected osteosyntheses, treated according to the ICS (Infection, Callus, Stability) classification in two Italian hospitals. Patients classified as ICS Type 1 (N = 83) feature callus progression and hardware stability, in spite of the presence of infection; these patients were treated with suppressive antibiotic therapy coupled with local debridement in 18.1% of the cases, and no hardware removal until bone healing. Type 2 patients (N = 75) are characterized by the presence of infection and hardware stability, but no callus progression; these patients were treated as Type 1 patients, but with additional callus stimulation therapies. Type 3 patients (N = 57), showing infection, no callus progression and loss of hardware stability, underwent removal and exchange of the fixation device. Considering only the initial treatment, performed according to the ICS classification, at a minimum 5 years follow up, 89.3% achieved bone healing and 93.5% did not show infection recurrence. The ICS classification appears as a useful and reliable tool to help standardizing the decision-making process in treating infected osteosynthesis with the most conservative approach. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biomechanics of far cortical locking.

PubMed

Bottlang, Michael; Feist, Florian

2011-02-01

The development of far cortical locking (FCL) was motivated by a conundrum: locked plating constructs provide inherently rigid stabilization, yet they should facilitate biologic fixation and secondary bone healing that relies on flexible fixation to stimulate callus formation. Recent studies have confirmed that the high stiffness of standard locked plating constructs can suppress interfragmentary motion to a level that is insufficient to reliably promote secondary fracture healing by callus formation. Furthermore, rigid locking screws cause an uneven stress distribution that may lead to stress fracture at the end screw and stress shielding under the plate. This review summarizes four key features of FCL constructs that have been shown to enhance fixation and healing of fractures: flexible fixation, load distribution, progressive stiffening, and parallel interfragmentary motion. Specifically, flexible fixation provided by FCL reduces the stiffness of a locked plating construct by 80% to 88% to actively promote callus proliferation similar to an external fixator. Load is evenly distributed between FCL screws to mitigate stress risers at the end screw. Progressive stiffening occurs by near cortex support of FCL screws and provides additional support under elevated loading. Finally, parallel interfragmentary motion by the S-shaped flexion of FCL screws promotes symmetric callus formation. In combination, these features of FCL constructs have been shown to induce more callus and to yield significantly stronger and more consistent healing compared with standard locked plating constructs. As such, FCL constructs function as true internal fixators by replicating the biomechanical behavior and biologic healing response of external fixators.

Biomechanics of Far Cortical Locking

PubMed Central

Bottlang, Michael; Feist, Florian

2011-01-01

The development of FCL was motivated by a conundrum: locked plating constructs provide inherently rigid stabilization, yet they should facilitate biological fixation and secondary bone healing that relies on flexible fixation to stimulate callus formation. Recent studies have confirmed that the high stiffness of standard locked plating constructs can suppress interfragmentary motion to a level that is insufficient to reliably promote secondary fracture healing by callus formation. Furthermore, rigid locking screws cause an uneven stress distribution that may lead to stress fracture at the end screw and stress shielding under the plate. This review summarizes four key features of FCL constructs that have shown to enhance fixation and fracture healing: Flexible fixation, load distribution, progressive stiffening, and parallel interfragmentary motion. Specifically, flexible fixation provided by FCL reduces the stiffness of a locked plating construct by 80–88% to actively promote callus proliferation similar to an external fixator. Load distribution is evenly shared between FCL screws to mitigate stress risers at the end screw. Progressive stiffening occurs by near cortex support of FCL screws and provides additional support under elevated loading. Finally, parallel interfragmentary motion by s-shaped flexion of FCL screws has shown to induce symmetric callus formation. In combination, these features of FCL constructs have shown to induce more callus and to yield significantly stronger and more consistent healing compared to standard locked plating constructs. As such, FCL constructs function as true internal fixators by replicating the biomechanical behavior and biological healing response of external fixators. PMID:21248556

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea.

PubMed

Prabhudesai, V; Bhaskaran, S

1993-03-01

An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL(-1) BA and 0.1 mgL(-1) NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL(-1)), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.

Sostdc1 deficiency accelerates fracture healing by promoting the expansion of periosteal mesenchymal stem cells [Sostdc1 Participates in Bone Maintenance and Fracture Repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collette, Nicole M.; Yee, Cristal S.; Hum, Nicholas R.

Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 ( Sostdc1 –/–) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1 –/– cortical bone measurements revealed larger bones with higher BMD,more » suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1 –/– mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1 –/– 5 day calluses harbor > 2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. In conclusion, these data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1 –/– mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum.« less

Appearance and accumulation of C/sub 4/ carbon pathway enzymes in developing maize leaves and differentiating maize A188 callus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoyagi, K.; Bassham, J.A.

1986-02-01

Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C/sub 4/ photosynthesis, for cell morphology, and for /sup 14/C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, ECmore » 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later. Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C/sub 3/ leaves and from etiolated C/sub 4/ leaf tissues, but differing from the corresponding enzymes in the mature leaf. Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology. Analysis of the labeled products of /sup 14/CO/sub 2/ fixation by nonchlorophyllous calli indicated ..beta..-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in ..beta..-carboxylation products was even greater.« less

Sostdc1 deficiency accelerates fracture healing by promoting the expansion of periosteal mesenchymal stem cells [Sostdc1 Participates in Bone Maintenance and Fracture Repair

DOE PAGES

Collette, Nicole M.; Yee, Cristal S.; Hum, Nicholas R.; ...

2016-04-19

Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 ( Sostdc1 –/–) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1 –/– cortical bone measurements revealed larger bones with higher BMD,more » suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1 –/– mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1 –/– 5 day calluses harbor > 2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. In conclusion, these data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1 –/– mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum.« less

In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant

PubMed Central

Sen, Monokesh Kumer; Nasrin, Shamima; Rahman, Shahedur; Jamal, Abu Hena Mostofa

2014-01-01

Objective To study callus induction from different explants (internode, leaf, root) and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L. Methods Sterilized explants were prepared by using 0.1% HgCl2 and 0.5% Bavistin and callus was obtained when cultured onto Murashige Skoog's (MS) medium by using different concentrations and combination of 2,4-D, NAA, BAP, IAA, IBA with 3% sucrose and 0.8% agar. Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively. Results Sterilization treatment of 0.1% HgCl2 for 2-3 min and Bavistin 0.5% for 10-12 min showed the highest percentage of asepsis and survival rate. Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf. Highest shootlets number (4.83±0.17) and length (3.8±0.16) cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L. Concerted efforts of BAP 2.0 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number (6.77±0.94). In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations. Experimentally, 3.0 mg/L IBA was enabled to induce maximum rootlets number (10.0±9.82) on full strength MS medium. Afterwards, regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized. The survived plantlets showed 66.67% survival frequency without any morphological abnormality. Conclusions The results demonstrated that different explants were good source of callus induction, morphology analysis as well as indirect plantlets regeneration. PMID:24144129

De novo assembly and comparative analysis of the transcriptome of embryogenic callus formation in bread wheat (Triticum aestivum L.).

PubMed

Chu, Zongli; Chen, Junying; Sun, Junyan; Dong, Zhongdong; Yang, Xia; Wang, Ying; Xu, Haixia; Zhang, Xiaoke; Chen, Feng; Cui, Dangqun

2017-12-19

During asexual reproduction the embryogenic callus can differentiate into a new plantlet, offering great potential for fostering in vitro culture efficiency in plants. The immature embryos (IMEs) of wheat (Triticum aestivum L.) are more easily able to generate embryogenic callus than mature embryos (MEs). To understand the molecular process of embryogenic callus formation in wheat, de novo transcriptome sequencing was used to generate transcriptome sequences from calli derived from IMEs and MEs after 3d, 6d, or 15d of culture (DC). In total, 155 million high quality paired-end reads were obtained from the 6 cDNA libraries. Our de novo assembly generated 142,221 unigenes, of which 59,976 (42.17%) were annotated with a significant Blastx against nr, Pfam, Swissprot, KOG, KEGG, GO and COG/KOG databases. Comparative transcriptome analysis indicated that a total of 5194 differentially expressed genes (DEGs) were identified in the comparisons of IME vs. ME at the three stages, including 3181, 2085 and 1468 DEGs at 3, 6 and 15 DC, respectively. Of them, 283 overlapped in all the three comparisons. Furthermore, 4731 DEGs were identified in the comparisons between stages in IMEs and MEs. Functional analysis revealed that 271transcription factor (TF) genes (10 overlapped in all 3 comparisons of IME vs. ME) and 346 somatic embryogenesis related genes (SSEGs; 35 overlapped in all 3 comparisons of IME vs. ME) were differentially expressed in at least one comparison of IME vs. ME. In addition, of the 283 overlapped DEGs in the 3 comparisons of IME vs. ME, excluding the SSEGs and TFs, 39 possessed a higher rate of involvement in biological processes relating to response to stimuli, in multi-organism processes, reproductive processes and reproduction. Furthermore, 7 were simultaneously differentially expressed in the 2 comparisons between the stages in IMEs, but not MEs, suggesting that they may be related to embryogenic callus formation. The expression levels of genes, which were validated by qRT-PCR, showed a high correlation with the RNA-seq value. This study provides new insights into the role of the transcriptome in embryogenic callus formation in wheat, and will serve as a valuable resource for further studies addressing embryogenic callus formation in plants.

Fracture healing in osteoporotic bone.

PubMed

Cheung, Wing Hoi; Miclau, Theodore; Chow, Simon Kwoon-Ho; Yang, Frank F; Alt, Volker

2016-06-01

As the world population rises, osteoporotic fracture is an emerging global threat to the well-being of elderly patients. The process of fracture healing by intramembranous ossification or/and endochondral ossification involve many well-orchestrated events including the signaling, recruitment and differentiation of mesenchymal stem cells (MSCs) during the early phase; formation of a hard callus and extracellular matrix, angiogenesis and revascularization during the mid-phase; and finally callus remodeling at the late phase of fracture healing. Through clinical and animal research, many of these factors are shown to be impaired in osteoporotic bone. Animal studies related to post-menopausal estrogen deficient osteoporosis (type I) have shown healing to be prolonged with decreased levels of MSCs and decreased levels of angiogenesis. Moreover, the expression of estrogen receptor (ER) was shown to be delayed in ovariectomy-induced osteoporotic fracture. This might be related to the observed difference in mechanical sensitivity between normal and osteoporotic bones, which requires further experiments to elucidate. In mice fracture models related to senile osteoporosis (type II), it was observed that chondrocyte and osteoblast differentiation were impaired; and that transplantation of juvenile bone marrow would result in enhanced callus formation. Other factors related to angiogenesis and vasculogenesis have also been noted to be impaired in aged models, affecting the degradation of cartilaginous matrixes and vascular invasion; the result is changes in matrix composition and growth factors concentrations that ultimately impairs healing during age-related osteoporosis. Most osteoporotic related fractures occur at metaphyseal sites clinically, and reports have indicated that differences exist between diaphyseal and metaphyseal fractures. An animal model that satisfies three main criteria (metaphyseal region, plate fixation, osteoporosis) is suggested for future research for more comprehensive understanding of the impairment in osteoporotic fractures. Therefore, a metaphyseal fracture or osteotomy that achieves complete discontinuity fixed with metal implants is suggested on ovariectomized aged rodent models. © 2016 Elsevier Ltd. All rights reserved.

Biotechnological experiments in space flights on board of space stations

NASA Astrophysics Data System (ADS)

Nechitailo, Galina S.

2012-07-01

Space flight conditions are stressful for any plant and cause structural-functional transition due to mobiliation of adaptivity. In space flight experiments with pea tissue, wheat and arabidopsis we found anatomical-morphological transformations and biochemistry of plants. In following experiments, tissue of stevia (Stevia rebaudiana), potato (Solanum tuberosum), callus culture and culture and bulbs of suffron (Crocus sativus), callus culture of ginseng (Panax ginseng) were investigated. Experiments with stevia carried out in special chambers. The duration of experiment was 8-14 days. Board lamp was used for illumination of the plants. After experiment the plants grew in the same chamber and after 50 days the plants were moved into artificial ionexchange soil. The biochemical analysis of plants was done. The total concentration of glycozides and ratio of stevioside and rebauside were found different in space and ground plants. In following generations of stevia after flight the total concentration of stevioside and rebauside remains higher than in ground plants. Experiments with callus culture of suffron carried out in tubes. Duration of space flight experiment was 8-167 days. Board lamp was used for illumination of the plants. We found picrocitina pigment in the space plants but not in ground plants. Tissue culture of ginseng was grown in special container in thermostate under stable temperature of 22 ± 0,5 C. Duration of space experiment was from 8 to 167 days. Biological activity of space flight culutre was in 5 times higher than the ground culture. This difference was observed after recultivation of space flight samples on Earth during year after flight. Callus tissue of potato was grown in tubes in thermostate under stable temperature of 22 ± 0,5 C. Duration of space experiment was from 8 to 14 days. Concentration of regenerates in flight samples was in 5 times higher than in ground samples. The space flight experiments show, that microgravity and other factors of space flight change direction of biological processes, and show a possibility to get special kinds of bioproducts with new properties.

Effect of autocontrol micromotion intramedullary interlocking nail on fracture healing: an experimental study.

PubMed

Xu, Wei-zhou; Guo, Xiao-dong; Zhao, Ju-cai; Wang, Yi-jin

2006-06-01

To investigate the effect of autocontrol micromotion locking nail (AMLN) on experimental fracture healing and its mechanism. 16 goats undergoing both sides of transverse osteotomy of the femoral shafts were fixed intramedullary with AMLN and Gross-Kempf (GK) nail, respectively. The follow-up time was 7, 14, 28 and 56 days. Roentgenographic, biomechanical, histological, scanning electromicroscopic and biochemical analyses were done. (1) The strength of anticompression, antiflexion and antitorsion in the fractural end in the AMLN-fixed group was higher than that of GK nail-fixed group; whereas, the rate of stress shelter in the fractured end decreased significantly (P<0.01). (2) The content of the total collagen, insoluble collagen, calcium and phosphate in the AMLN-fixed group was higher than that in the GK nail-fixed group (P<0.05). (3) Histological observation and quantitative analysis of calluses revealed that AMLN could promote the growth of bridge calluses and periosteum calluses. Hence the fracture healing and remolding process achieved early, which was much better than traditional GK nail fixation. (P<0.05). (4) 7-14 days postoperation, the calluses of AMLN-fixed group was flourish and camellarly arranged and the collagen fibril formed constantly in the absorption lacuna of bone trabecula. 28-56 days postoperation, the collagen fibril was flourish around the absorption lacuna and was parallel to the bone's longitudinal axis. Active bony absorption and formation were seen, so was remolding and rebuilding. Haversian system was intact and the bony structural net was very tenacious because of the deposition of calcium salt. None of the above findings was observed in the GK nail-fixed group. The design of AMLN accords well with the plastic fixation theory. As the geometry ametabolic system constituted by the intramedullary fixation instruments and the proximal and distal end of the fracture is very firm and stable, the disturbance to the physical stress distributed in the fractural end is light. The generation and conduct of the intermittent physical stress between the fractural parts could reach the balance between stress conduct and stress protection. The feature that the healing and remolding take place at the same time speeds up the fractural healing process.

Molecular Transducers from Roots Are Triggered in Arabidopsis Leaves by Root-Knot Nematodes for Successful Feeding Site Formation: A Conserved Post-Embryogenic De novo Organogenesis Program?

PubMed Central

Olmo, Rocío; Cabrera, Javier; Moreno-Risueno, Miguel A.; Fukaki, Hidehiro; Fenoll, Carmen; Escobar, Carolina

2017-01-01

Root-knot nematodes (RKNs; Meloidogyne spp.) induce feeding cells (giant cells; GCs) inside a pseudo-organ (gall) from still unknown root cells. Understanding GCs ontogeny is essential to the basic knowledge of RKN–plant interaction and to discover novel and effective control strategies. Hence, we report for the first time in a model plant, Arabidopsis, molecular, and cellular features concerning ectopic de novo organogenesis of RKNs GCs in leaves. RKNs induce GCs in leaves with irregular shape, a reticulated cytosol, and fragmented vacuoles as GCs from roots. Leaf cells around the nematode enter G2-M shown by ProCycB1;1:CycB1;1(NT)-GUS expression, consistent to multinucleated GCs. In addition, GCs nuclei present irregular and varied sizes. All these characteristics mentioned, being equivalent to GCs in root-galls. RKNs complete their life cycle forming a gall/callus-like structure in the leaf vascular tissues resembling auxin-induced callus with an auxin-response maxima, indicated by high expression of DR5::GUS that is dependent on leaf auxin-transport. Notably, induction of leaves calli/GCs requires molecular components from roots crucial for lateral roots (LRs), auxin-induced callus and root-gall formation, i.e., LBD16. Hence, LBD16 is a xylem pole pericycle specific and local marker in LR primordia unexpectedly induced locally in the vascular tissue of leaves after RKN infection. LBD16 is also fundamental for feeding site formation as RKNs could not stablish in 35S::LBD16-SRDX leaves, and likely it is also a conserved molecular hub between biotic and developmental signals in Arabidopsis either in roots or leaves. Moreover, RKNs induce the ectopic development of roots from leaf and root-galls, also formed in mutants compromised in LR formation, arf7/arf19, slr, and alf4. Therefore, nematodes must target molecular signatures to induce post-embryogenic de novo organogenesis through the LBD16 callus formation pathway partially different from those prevalent during normal LR development. PMID:28603536

Molecular Transducers from Roots Are Triggered in Arabidopsis Leaves by Root-Knot Nematodes for Successful Feeding Site Formation: A Conserved Post-Embryogenic De novo Organogenesis Program?

PubMed

Olmo, Rocío; Cabrera, Javier; Moreno-Risueno, Miguel A; Fukaki, Hidehiro; Fenoll, Carmen; Escobar, Carolina

2017-01-01

Root-knot nematodes (RKNs; Meloidogyne spp.) induce feeding cells (giant cells; GCs) inside a pseudo-organ (gall) from still unknown root cells. Understanding GCs ontogeny is essential to the basic knowledge of RKN-plant interaction and to discover novel and effective control strategies. Hence, we report for the first time in a model plant, Arabidopsis, molecular, and cellular features concerning ectopic de novo organogenesis of RKNs GCs in leaves. RKNs induce GCs in leaves with irregular shape, a reticulated cytosol, and fragmented vacuoles as GCs from roots. Leaf cells around the nematode enter G2-M shown by ProCycB1;1:CycB1;1(NT)-GUS expression, consistent to multinucleated GCs. In addition, GCs nuclei present irregular and varied sizes. All these characteristics mentioned, being equivalent to GCs in root-galls. RKNs complete their life cycle forming a gall/callus-like structure in the leaf vascular tissues resembling auxin-induced callus with an auxin-response maxima, indicated by high expression of DR5::GUS that is dependent on leaf auxin-transport. Notably, induction of leaves calli/GCs requires molecular components from roots crucial for lateral roots (LRs), auxin-induced callus and root-gall formation, i.e., LBD16. Hence, LBD16 is a xylem pole pericycle specific and local marker in LR primordia unexpectedly induced locally in the vascular tissue of leaves after RKN infection. LBD16 is also fundamental for feeding site formation as RKNs could not stablish in 35S::LBD16-SRDX leaves, and likely it is also a conserved molecular hub between biotic and developmental signals in Arabidopsis either in roots or leaves. Moreover, RKNs induce the ectopic development of roots from leaf and root-galls, also formed in mutants compromised in LR formation, arf7/arf19 , slr , and alf4 . Therefore, nematodes must target molecular signatures to induce post-embryogenic de novo organogenesis through the LBD16 callus formation pathway partially different from those prevalent during normal LR development.

Protocols for Callus and Somatic Embryo Initiation for Hibiscus sabdariffa L. (Malvaceae): Influence of Explant Type, Sugar, and Plant Growth Regulators

USDA-ARS?s Scientific Manuscript database

A significant work on callus induction and somatic embryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, c...

Use of culture filtrates of Ceratocystis ulmi as a bioassay to screen for disease tolerant Ulmus americana

Treesearch

Paula M. Pijut; Subash C. Domir; R. Daniel Lineberger; Lawrence R. Schreiber

1990-01-01

Callus cultures of elm (Ulmus americana L.) derived from Dutch elm disease susceptible, intermediate-resistant, and resistant genotypes were exposed to the culture filtrates of three pathogenic isolates of Ceratocystis ulmi, the causal agent of Dutch elm disease. Callus fresh weights, cell viability, and reactions of stem cuttings...

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures.

PubMed

Centeno, M L; Rodríguez, A; Feito, I; Fernández, B

1996-11-01

Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.

Allelopathy of small everlasting (Antennaria microphylla) : Phytotoxicity to leafy spurge (Euphorbia esula) in tissue culture.

PubMed

Hogan, M E; Manners, G D

1990-03-01

Media and media extracts from callus cultures of small everlasting (Antennaria microphylla) inhibited leafy spurge (Euphorbia esula L.) callus tissue and suspension culture growth (50 and 70% of control, respectively) and were phytotoxic in lettuce and leafy spurge root elongation bioassays (64 and 77% of control, respectively). Hydroquinone, a phytotoxic compound previously isolated from small everlasting, was also biosynthesized by callus and suspension cultures of this species. Exogenously supplied hydroquinone (0.5 mM) was toxic to leafy spurge suspension culture cells and was only partially biotransformed to its nontoxic water-soluble monoglucoside, arbutin, by these cells. This report confirms the chronic involvement of hydroquinone in the allelopathic interaction between small everlasting and leafy spurge.

Green and non-green callus induction from excised rice (Oryza sativa) embryos: effects of exogenous plant growth regulators

NASA Technical Reports Server (NTRS)

Kim, D.; Brock, T. G.; Kaufman, P. B.

1992-01-01

Calli were induced either from excised rice embryos or from whole seeds in the presence of 1 to 5 mg l-1 NAA. After 12 days of culture, calli were induced only from excised rice embryos. We found that excised embryos accumulated NAA up to 6 times higher concentration than did whole seeds. In the presence of 1 to 5 mg l-1 NAA and 2 to 10 mg l-1 kinetin, chlorophyllous calli were induced from excised rice embryos. Chlorophyll contents in the callus tissue increased with increasing kinetin concentration while percent callus induction decreased. The total chlorophyll content was linearly correlated with the ratio of kinetin to NAA in the medium.

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars.

PubMed

Basnayake, Shiromani W V; Moyle, Richard; Birch, Robert G

2011-03-01

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11-15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.

Effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia rebaudiana for Steviol glycoside production.

PubMed

Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

2014-03-01

Steviol glycosides are natural non-caloric sweeteners which are extracted from the leaves of Stevia rebaudiana plant. Present study deals the effect of salts (NaCl and Na2CO3) on callus and suspension culture of Stevia plant for steviol glycoside (SGs) production. Yellow-green and compact calli obtained from in vitro raised Stevia leaves sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of NaCl (0.05-0.20%) and Na2CO3 (0.0125-0.10%) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension biomass cultured on salts showed less growth as well as browning of medium when compared with control. Quantification of SGs content in callus culture (collected on 15th day) and suspension cultures (collected at 10th and 15th days) treated with and without salts were analyzed by HPLC. It was found that abiotic stress induced by the salts increased the concentration of SGs significantly. In callus, the quantity of SGs got increased from 0.27 (control) to 1.43 and 1.57% with 0.10% NaCl, and 0.025% Na2CO3, respectively. However, in case of suspension culture, the same concentrations of NaCl and Na2CO3 enhanced the SGs content from 1.36 (control) to 2.61 and 5.14%, respectively, on the 10th day.

Deferoxamine restores callus size, mineralization, and mechanical strength in fracture healing after radiotherapy.

PubMed

Donneys, Alexis; Ahsan, Salman; Perosky, Joseph E; Deshpande, Sagar S; Tchanque-Fossuo, Catherine N; Levi, Benjamin; Kozloff, Ken M; Buchman, Steven R

2013-05-01

Therapeutic augmentation of fracture-site angiogenesis with deferoxamine has proven to increase vascularity, callus size, and mineralization in long-bone fracture models. The authors posit that the addition of deferoxamine would enhance pathologic fracture healing in the setting of radiotherapy in a model where nonunions are the most common outcome. Thirty-five Sprague-Dawley rats were divided into three groups. Fracture, irradiated fracture, and irradiated fracture plus deferoxamine. The irradiated fracture and irradiated fracture plus deferoxamine groups received a human equivalent dose of radiotherapy [7 Gy/day for 5 days, (35 Gy)] 2 weeks before mandibular osteotomy and external fixation. The irradiated fracture plus deferoxamine group received injections of deferoxamine into the fracture callus after surgery. After a 40-day healing period, mandibles were dissected, clinically assessed for bony union, imaged with micro-computed tomography, and tension tested to failure. Compared with irradiated fractures, metrics of callus size, mineralization, and strength in deferoxamine-treated mandibles were significantly increased. These metrics were restored to a level demonstrating no statistical difference from control fractures. In addition, the authors observed an increased rate of achieving bony unions in the irradiated fracture plus deferoxamine-treated group when compared with irradiated fracture (67 percent and 20 percent, respectively). The authors' data demonstrate nearly total restoration of callus size, mineralization, and biomechanical strength, and a threefold increase in the rate of union with the use of deferoxamine. The authors' results suggest that the administration of deferoxamine may have the potential for clinical translation as a new treatment paradigm for radiation-induced pathologic fractures.

High Triterpenic Acids Production in Callus Cultures from Fruit Pulp of Two Apple Varieties.

PubMed

Verardo, Giancarlo; Gorassini, Andrea; Ricci, Donata; Fraternale, Daniele

2017-01-01

Very rarely fruit pulp has been used in in vitro culture to produce secondary metabolites useful in promoting health. The aims of this work were the study of the best conditions to obtain the callus cultures from the pulp of two varieties of apples, Golden Delicious (GD) and "Mela Rosa Marchigiana" (MRM), and the quali-quantitative analysis of secondary metabolites produced by the two in vitro callus cultures. Callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of supplements. To achieve the maximum recovery of secondary metabolites produced, preliminary extraction tests were carried out on GD apple culture using two different organic solvents (MeOH and EtOAc). The quali-quantitative analysis of the methanolic extract of both cultures was carried out by ESI-MS n and GC-MS techniques. The GC-MS analysis revealed the presence of triterpenic acids, in particular, oleanolic, ursolic, maslinic, pomolic, tormentic, corosolic and annurcoic acid along with a phytosterol, β-sitosterol. In addition, GD callus culture produced phloridzin, absent in the MRM culture. In this last culture, however, the total amount of secondary metabolites was markedly higher. The in vivo production of these bioactive compounds were also quantified in the GD and MRM apple pulps. Apple pulps produced higher amounts of triterpenic acids in vitro than in vivo. The present work can be considered a method to amplify the production of important secondary metabolites which exert beneficial effects on human health. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Exogenous PTHrP Repairs the Damaged Fracture Healing of PTHrP+/− Mice and Accelerates Fracture Healing of Wild Mice

PubMed Central

Wang, Yinhe; Fang, Xin; Wang, Chun; Ding, Congzhu; Lin, Hua; Liu, Anlong; Wang, Lei; Cao, Yang

2017-01-01

Bone fracture healing is a complicated physiological regenerative process initiated in response to injury and is similar to bone development. To demonstrate whether an exogenous supply of parathyroid hormone–related protein (PTHrP) helps in bone fracture healing, closed mid-diaphyseal femur fractures were created and stabilized with intramedullary pins in eight-week-old wild-type (WT) PTHrP+/+ and PTHrP+/− mice. After administering PTHrP for two weeks, callus tissue properties were analyzed at one, two, and four weeks post-fracture (PF) by various methods. Bone formation–related genes and protein expression levels were evaluated by real-time reverse transcriptase–polymerase chain reaction and Western blots. At two weeks PF, mineral density of callus, bony callus areas, mRNA levels of alkaline phosphatase (ALP), type I collagen, Runt-related transcription factor 2 (Runx-2), and protein levels of Runx-2 and insulin-like growth factor-1 decreased in PTHrP+/− mice compared with WT mice. At four weeks PF, total collagen-positive bony callus areas, osteoblast number, ALP-positive areas, and type I collagen-positive areas all decreased in PTHrP+/− mice. At both two and four weeks PF, tartrate-resistant acid phosphatase–positive osteoclast number and surface decreased a little in PTHrP+/− mice. The study indicates that exogenous PTHrP provided by subcutaneous injection could redress impaired bone fracture healing, leading to mutation of activated PTHrP by influencing callus areas, endochondral bone formation, osteoblastic bone formation, and bone turnover. PMID:28178186

21 CFR 358.550 - Labeling of corn and callus remover drug products.

Code of Federal Regulations, 2010 CFR

2010-04-01

... formulated in a collodion-like vehicle. (i) “If product gets into the eye, flush with water for 15 minutes...(a). “Wash affected area and dry thoroughly.” (If appropriate: “Cut plaster to fit corn/callus... in § 358.510(b). “Wash affected area and dry thoroughly. Apply” (select one of the following, as...

21 CFR 358.550 - Labeling of corn and callus remover drug products.

Code of Federal Regulations, 2012 CFR

2012-04-01

... formulated in a collodion-like vehicle. (i) “If product gets into the eye, flush with water for 15 minutes...(a). “Wash affected area and dry thoroughly.” (If appropriate: “Cut plaster to fit corn/callus... in § 358.510(b). “Wash affected area and dry thoroughly. Apply” (select one of the following, as...

21 CFR 358.550 - Labeling of corn and callus remover drug products.

Code of Federal Regulations, 2014 CFR

2014-04-01

... formulated in a collodion-like vehicle. (i) “If product gets into the eye, flush with water for 15 minutes...(a). “Wash affected area and dry thoroughly.” (If appropriate: “Cut plaster to fit corn/callus... in § 358.510(b). “Wash affected area and dry thoroughly. Apply” (select one of the following, as...

21 CFR 358.550 - Labeling of corn and callus remover drug products.

Code of Federal Regulations, 2011 CFR

2011-04-01

... formulated in a collodion-like vehicle. (i) “If product gets into the eye, flush with water for 15 minutes...(a). “Wash affected area and dry thoroughly.” (If appropriate: “Cut plaster to fit corn/callus... in § 358.510(b). “Wash affected area and dry thoroughly. Apply” (select one of the following, as...

21 CFR 358.550 - Labeling of corn and callus remover drug products.

Code of Federal Regulations, 2013 CFR

2013-04-01

... formulated in a collodion-like vehicle. (i) “If product gets into the eye, flush with water for 15 minutes...(a). “Wash affected area and dry thoroughly.” (If appropriate: “Cut plaster to fit corn/callus... in § 358.510(b). “Wash affected area and dry thoroughly. Apply” (select one of the following, as...

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Treesearch

Mohammad-Shafie Rahmani; Paula M. Pijut; Naghi Shabanian

2016-01-01

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability;...

Solid-stemmed spring wheat cultivars give better androgenic response than hollow-stemmed cultivars in anther culture.

PubMed

Weigt, Dorota; Kiel, Angelika; Nawracała, Jerzy; Pluta, Mateusz; Łacka, Agnieszka

2016-01-01

Solid-stemmed spring wheat cultivars ( Triticum aestivum L.) are resistant to the stem sawfly ( Cephus cinctus Nort.) and lodging. Anthers of 24 spring wheat cultivars with varying content of pith in the stem were used in the experiment. All were classified into three groups: solid, medium-solid and hollow stems. There was considerable influence of the cultivar on callus formation and green plant regeneration. The highest efficiency of green plant regeneration (24%) was observed for the solid-stemmed AC Abbey cultivar. There was no regeneration from the explants of four cultivars: CLTR 7027, Alentejano, Marquis and Bombona. Principal component analysis showed no differences between the cases under observation (callus induction and green plant regeneration) in their response to pre-treatment temperatures (4 and 8°C). The examination of the effects of various auxin types in the induction medium on callus formation and green plant regeneration revealed that the strongest stimulation of these processes was observed in the C17 medium with 2,4-D and dicamba. The efficiency of callus formation and green plant regeneration was greater in solid-stemmed cultivars than in hollow-stemmed cultivars.

Effects of Salinity on growth and osmotic regulation substances of callus induced from Reaumuria soongorica

NASA Astrophysics Data System (ADS)

Tan, Huijuan; Li, Xinrong; Liu, Yubing; Zhao, Xin

2014-05-01

Reaumuria soongorica (Pall.) Maxim is the strong xerophils plant in the northwest arid and semiarid regions in China. It plays very important roles in stabilizing sand dunes and in construction of agricultural shelter belts in north-west China.The present study aimed to evaluate the response to salinity of R. soongorica, which is more salt-resistant than other valuable shrub species used for afforestation on saline and alkaline desert, at the cellular level. To this purpose, callus was induced from shoot segments of R. soongorica on Murashige and Skoog (MS) medium supplemented with 0.2 mgL-16-benzyladenine (BA) and 2.0 mg mgL-1 2,4-Dichlorophenoxyacetic acid (2 ,4-D). The relative growth rate of callus reached a maximum in the presence of 100 mmol L-1NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, trehalose, Glycine betain and flavonoids increased with increasing salt concentrations. The results indicate that the response of the callus of R. soongorica to salt stress is similar to that of the whole plant. .

[Introduction of hexaploid of Chinese narcissus and analysis of its chromosome change].

PubMed

Wang, Rui; Zhang, Ya Nan; Wang, Ya Ying; Tian, Hui Qiao

2007-06-01

Anthers of Chinese narcissus (Narcissus tazetta L. var chinesis Roem) were used as explants for callus induction and plant regeneration. About 80% anthers produced callus and 28% of the callus differentiated out bulbs, making a good experiment system of tissue culture of Chinese narcissus for further cellular and gene engineering. The 700 callus were treated by 0.5% colchicin for 5-6 days and then transformed into a MS medium containing 3 mg/L 6-BA to induce differentiation. 90 bulbs were obtained and 55 bulbs among them were checked the chromosome number from their root tips for three times. 29 bulbs (53%, 29/55) still kept triploidy and the most cells of root tips contained 30 chromosomes. 22 bulbs (40%, 22/55) displayed aneuploidy and the most cells of its root tips contained 10-50 chromosomes. 4 bulbs displayed hexaploidy and contained 60 chromosomes. After three months growing, the cells of root tips containing aneuploidy chromosomes disappeared, and the bulbs became triploidy. The chromosomes of 4 hexaploidy bulbs did not changed during three checks. The origin and disappearance of aneuploidy cells of Chinese narcissus after treated by colchicin were discussed.

[Chemistry and biosynthesis of prenylflavonoids].

PubMed

Nomura, T

2001-07-01

Many isoprenylated flavonoids have been isolated from mulberry trees and related plants (Moraceae). Among them, kuwanons G (13) and H (14) were the first isolated active substances exhibiting a hypotensive effect from the Japanese Morus root bark. These compounds are considered to be formed through an enzymatic Diels-Alder reaction of a chalcone (15) and dehydro-kuwanon C (16) or its equivalent. Since that time, about forty kinds of Diels-Alder type adducts structurally similar to that of 13 have been isolated from the moraceous plants. Some strains of Morus alba as well as M. bombycis callus tissues have a high productivity of mulberry Diels-Alder type adducts, such as chalcomoracin (26) and kuwanon J (28). The biosynthesis of the mulberry Diels-Alder type adducts has been studied with the aid of the cell strain. Chalcomoracin (26) and kuwanon J (28) were proved to be enzymatic Diels-Alder type reaction products by the administration experiment with O-methylchalcone derivatives. Furthermore, for the isoprenoid biosynthesis of prenylflavonoids in Morus alba callus tissues, a novel way through the junction of glycolysis and pentose-phosphate cycle was proposed. The crude enzyme fraction catalyzing the Morus Diels-Alder type reaction could be isolated. Studies of phenolic constituents of licorice (Glycyrrhiza species) were carried out. On the course of the structure determination of the phenolic constituents of licorice, two new NMR structure determination methods for prenylflavonoids were found. Furthermore, the prenylphenols isolated from licorice were summarized according to the origin of the materials.

A new approach for in vitro regeneration of tomato plants devoid of exogenous plant growth hormones.

PubMed

Plana, Dagmara; Fuentes, Alejandro; Alvarez, Marta; Lara, Regla M; Alvarez, Félix; Pujol, Merardo

2006-10-01

Many available methodologies for in vitro regeneration of commercial tomato varieties promote not only the production of normal shoots but also individual leaves, shoots without apical meristems and vitrified structures. All these abnormal formations influence and diminish the regeneration efficiency. At the basis of this phenomenon lies callus development. We optimized an alternative procedure by which the regeneration occurs without abnormal shoot formation. The portion including the proximal part of hypocotyls and the radicle was cultured on medium consisting of Murashige and Skoog salts, 4 mg/L thiamine, 100 mg/L mio-inositol and 3% sucrose. After two-three weeks, 60% explants showed adventitious shoot formation. No changes in the morphological characteristics of regenerated plants and fruits were observed as compared with parents. Karyotypic analysis of regenerated plants showed no variations in chromosome number. The optimized procedure offers the advantage of tomato plant regeneration avoiding callus formation, which enables normal plant recovery with an efficiency ranging from 1.45 +/- 0.05 to 2.57 +/- 0.06 shoots per explant in Campbell-28, Amalia, Lignon, and Floradel cultivars.

Micropropagation and non-steroidal anti-inflammatory and anti-arthritic agent boswellic acid production in callus cultures of Boswellia serrata Roxb.

PubMed

Nikam, Tukaram D; Ghorpade, Ravi P; Nitnaware, Kirti M; Ahire, Mahendra L; Lokhande, Vinayak H; Chopra, Arvind

2013-01-01

Micropropagation through cotyledonary and leaf node and boswellic acid production in stem callus of a woody medicinal endangered tree species Boswellia serrata Roxb. is reported. The response for shoots, roots and callus formation were varied in cotyledonary and leafy nodal explants from in vitro germinated seeds, if inoculated on Murshige and Skoog's (MS) medium fortified with cytokinins and auxins alone or together. A maximum of 8.0 ± 0.1 shoots/cotyledonary node explant and 6.9 ± 0.1 shoots/leafy node explants were produced in 91 and 88 % cultures respectively on medium with 2.5 μM 6-benzyladenine (BA) and 200 mg l(-1) polyvinylpyrrolidone (PVP). Shoots treated with 2.5 μM IBA showed the highest average root number (4.5) and the highest percentage of rooting (89 %). Well rooted plantlets were acclimatized and 76.5 % of the plantlets showed survival upon transfer to field conditions. Randomly amplified polymorphic DNA (RAPD) analysis of the micropropagated plants compared with mother plant revealed true-to-type nature. The four major boswellic acid components in calluses raised from root, stem, cotyledon and leaf explants were analyzed using HPLC. The total content of four boswellic acid components was higher in stem callus obtained on MS with 15.0 μM IAA, 5.0 μM BA and 200 mg l(-1) PVP. The protocol reported can be used for conservation and exploitation of in vitro production of medicinally important non-steroidal anti-inflammatory metabolites of B. serrata.

Site Specific Effects of Zoledronic Acid during Tibial and Mandibular Fracture Repair

PubMed Central

Yu, Yan Yiu; Lieu, Shirley; Hu, Diane; Miclau, Theodore; Colnot, Céline

2012-01-01

Numerous factors can affect skeletal regeneration, including the extent of bone injury, mechanical loading, inflammation and exogenous molecules. Bisphosphonates are anticatabolic agents that have been widely used to treat a variety of metabolic bone diseases. Zoledronate (ZA), a nitrogen-containing bisphosphonate (N-BP), is the most potent bisphosphonate among the clinically approved bisphosphonates. Cases of bisphosphonate-induced osteonecrosis of the jaw have been reported in patients receiving long term N-BP treatment. Yet, osteonecrosis does not occur in long bones. The aim of this study was to compare the effects of zoledronate on long bone and cranial bone regeneration using a previously established model of non-stabilized tibial fractures and a new model of mandibular fracture repair. Contrary to tibial fractures, which heal mainly through endochondral ossification, mandibular fractures healed via endochondral and intramembranous ossification with a lesser degree of endochondral ossification compared to tibial fractures. In the tibia, ZA reduced callus and cartilage formation during the early stages of repair. In parallel, we found a delay in cartilage hypertrophy and a decrease in angiogenesis during the soft callus phase of repair. During later stages of repair, ZA delayed callus, cartilage and bone remodeling. In the mandible, ZA delayed callus, cartilage and bone remodeling in correlation with a decrease in osteoclast number during the soft and hard callus phases of repair. These results reveal a more profound impact of ZA on cartilage and bone remodeling in the mandible compared to the tibia. This may predispose mandible bone to adverse effects of ZA in disease conditions. These results also imply that therapeutic effects of ZA may need to be optimized using time and dose-specific treatments in cranial versus long bones. PMID:22359627

Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: Effects of light and auxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lawyer, A.L.; Grady, K.L.; Bassham, J.A.

1981-10-01

Callus cultures derived from pith tissue of Nicotiana tobacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrams per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing (U-/sup 14/C)sucrose. The dry weight proportions of the callimore » (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increase 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH/sub 3/ content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) (U-/sup 14/C)sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeling glucose and fructose. Respiration of /sup 14/CO/sub 2/ followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.« less

Biomass Yield and Steviol Glycoside Production in Callus and Suspension Culture of Stevia rebaudiana Treated with Proline and Polyethylene Glycol.

PubMed

Gupta, Pratibha; Sharma, Satyawati; Saxena, Sanjay

2015-06-01

Enhanced production of steviol glycosides (SGs) was observed in callus and suspension culture of Stevia rebaudiana treated with proline and polyethylene glycol (PEG). To study their effect, yellow-green and compact calli obtained from in vitro raised Stevia leaves were sub-cultured on MS medium supplemented with 2.0 mg l(-1) NAA and different concentrations of proline (2.5-10 mM) and PEG (2.5-10 %) for 2 weeks, and incubated at 24 ± 1 °C and 22.4 μmol m(-2) s(-1) light intensity provided by white fluorescent tubes for 16 h. Callus and suspension culture biomass (i.e. both fresh and dry weight content) was increased with 5 mM proline and 5 % PEG, while at further higher concentrations, they got reduced. Further, quantification of SGs content in callus (collected at 15th day) and suspension culture (collected at 10th and 15th day) treated with and without elicitors was analysed by HPLC. It was observed that chemical stress enhanced the production of SGs significantly. In callus, the content of SGs increased from 0.27 (control) to 1.09 and 1.83 % with 7.5 mM proline and 5 % PEG, respectively, which was about 4.0 and 7.0 times higher than control. However, in the case of suspension culture, the same concentrations of proline and polyethylene glycol enhanced the SG content from 1.36 (control) to 5.03 and 6.38 %, respectively, on 10th day which were 3.7 times and 4.7 times higher than control.

Simulation of peri-implant bone healing due to immediate loading in dental implant treatments.

PubMed

Chou, Hsuan-Yu; Müftü, Sinan

2013-03-15

The goal of this work was to investigate the role of immediate loading on the peri-implant bone healing in dental implant treatments. A mechano-regulatory tissue differentiation model that takes into account the stimuli through the solid and the fluid components of the healing tissue, and the diffusion of pluripotent stem cells into the healing callus was used. A two-dimensional axisymmetric model consisting of a dental implant, the healing callus tissue and the host bone tissue was constructed for the finite element analysis. Poroelastic material properties were assigned to the healing callus and the bone tissue. The effects of micro-motion, healing callus size, and implant thread design on the length of the bone-to-implant contact (BIC) and the bone volume (BV) formed in the healing callus were investigated. In general, the analysis predicted formation of a continuous layer of soft tissue along the faces of the implant which are parallel to the loading direction. This was predicted to be correlated with the high levels of distortional strain transferred through the solid component of the stimulus. It was also predicted that the external threads on the implant, redistribute the interfacial load, thus help reduce the high distortional stimulus and also help the cells to differentiate to bone tissue. In addition, the region underneath the implant apex was predicted to experience high fluid stimulus that results in the development of soft tissue. The relationship between the variables considered in this study and the outcome measures, BV and BIC, was found to be highly nonlinear. A three-way analysis of variance (ANOVA) of the results was conducted and it showed that micro-motion presents the largest hindrance to bone formation during healing. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fracture healing with alendronate treatment in the Brtl/+ mouse model of osteogenesis imperfecta

PubMed Central

Meganck, J.A.; Begun, D.L.; McElderry, J.D.; Swick, A.; Kozloff, K.M.; Goldstein, S.A.; Morris, M.D.; Marini, J.C.; Caird, M.S.

2014-01-01

Osteogenesis imperfecta (OI) is a heritable bone dysplasia characterized by increased skeletal fragility. Patients are often treated with bisphosphonates to attempt to reduce fracture risk. However, bisphosphonates reside in the skeleton for many years and long-term administration may impact bone material quality. Acutely, there is concern about risk of non-union of fractures that occur near the time of bisphosphonate administration. This study investigated the effect of alendronate, a potent aminobisphosphonate, on fracture healing. Using the Brtl/+ murine model of type IV OI, tibial fractures were generated in 8-week-old mice that were untreated, treated with alendronate before fracture, or treated before and after fracture. After 2, 3, or 5 weeks of healing, tibiae were assessed using microcomputed tomography (μCT), torsion testing, quantitative histomorphometry, and Raman microspectroscopy. There were no morphologic, biomechanical or histomorphometric differences in callus between untreated mice and mice that received alendronate before fracture. Alendronate treatment before fracture did not cause a significant increase in cartilage retention in fracture callus. Both Brtl/+ and WT mice that received alendronate before and after fracture had increases in the callus volume, bone volume fraction and torque at failure after 5 weeks of healing. Raman microspectroscopy results did not show any effects of alendronate in wild-type mice, but calluses from Brtl/+ mice treated with alendronate during healing had a decreased mineral-to-matrix ratio, decreased crystallinity and an increased carbonate-to-phosphate ratio. Treatment with alendronate altered the dynamics of healing by preventing callus volume decreases later in the healing process. Fracture healing in Brtl/+ untreated animals was not significantly different from animals in which alendronate was halted at the time of fracture. PMID:23774443

Elicitation of Medicinally Important Antioxidant Secondary Metabolites with Silver and Gold Nanoparticles in Callus Cultures of Prunella vulgaris L.

PubMed

Fazal, Hina; Abbasi, Bilal Haider; Ahmad, Nisar; Ali, Mohammad

2016-11-01

Prunella vulgaris L. (P. vulgaris) is an important medicinal plant with a wide range of antiviral properties. Traditionally, it is known as self-heal because of its faster effects on wound healing. It is commonly known as a natural antiseptic due to the presence of various polyphenols. There is lack of research efforts on its propagation and production of bioactive compounds under field and in vitro conditions. In this study, the effects of different ratios (1:2, 1:3, 2:1, and 3:1) of silver (Ag) and gold (Au) nanoparticles (NPs) alone or in combination with naphthalene acetic acid (NAA) were investigated for callus culture development and production of secondary metabolites. The Ag (30 μg l -1 ), AgAu (1:2), and AgAu (2:1) NPs in combination with NAA (2.0 mg l -1 ) enhanced callus proliferation (100 %) as compared to the control (95 %). Among the different NPs tested, AuNPs with or without NAA produced higher biomass in log phases (35-42 days) of growth kinetics. Furthermore, AgAu (1:3) and AuNPs alone enhanced total protein content (855 μg-BSAE/mg-fresh weight (FW)), superoxide dismutase (0.54 nM/min/mg-FW), and peroxidase (0.39 nM/min/mg-FW) enzymes in callus cultures. The AgAuNPs (1:3) in combination with NAA induced maximum accumulation of phenolics (TPC 9.57 mg/g-dry weight (DW)) and flavonoid (6.71 mg/g-DW) content. Moreover, AgAuNPs (3:1) without NAA enhanced antioxidant activity (87.85 %). This study provides the first evidence of NP effect on callus culture development and production of natural antioxidants in P. vulgaris.

Proteomic alterations in root tips of Arabidopsis thaliana seedlings under altered gravity conditions

NASA Astrophysics Data System (ADS)

Zheng, H. Q.; Wang, H.

Gravity has a profound influence on plant growth and development Removed the influence of gravitational acceleration by spaceflight caused a wide range of cellular changes in plant Whole seedling that germinated and grown on clinostats showed the absent of gravitropism At the cellular level clinostat treatment has specific effects on plant cells such as induce alterations in cell wall composition increase production of heat-soluble proteins impact on the cellular energy metabolism facilitate a uniform distribution of plastids amyloplasts and increase number and volume of nucleoli A number of recent studies have shown that the exposure of Arabidopsis seedlings and callus cells to gravity stimulation hyper g-forces or clinostat rotation induces alterations in gene expression In our previous study the proteome of the Arabidopsis thaliana callus cells were separated by high resolution two-dimensional electrophoresis 2-DE Image analysis revealed that 80 protein spots showed quantitative and qualitative variations after exposure to clinostat rotation treatment We report here a systematic proteomic approach to investigate the altered gravity responsive proteins in root tip of Arabidopsis thaliana cv Landsberg erecta Three-day-old seedlings were exposed for 12h to a horizontal clinostat rotation H simulated weightlessness altered g-forces by centrifugation 7g hypergravity a vertical clinostat rotation V clinostat control or a stationary control grown conditions Total proteins of roots were extracted

Green synthesis of silver nanoparticles using Stevia leaves extracts

NASA Astrophysics Data System (ADS)

Laguta, Iryna; Stavinskaya, Oksana; Kazakova, Olga; Fesenko, Tetiana; Brychka, Sergey

2018-02-01

Three extracts of Stevia rebaudiana (Bertoni) were prepared using different types of raw materials: leaves of plants grown ex situ, leaves of plants grown in vitro, callus culture formed on damaged leaves. Composition of the extracts was studied by means of high-performance liquid chromatography and laser desorption/ionization mass spectrometry; total phenol content was estimated using Folin-Ciocalteau method. Flavonoids and hydroxycinnamic acids were found to be the main groups of phenol antioxidants available in the Stevia leaves, with the amount of these compounds in the extract being dependent on the type of raw material. The reducing properties of phenol compounds identified in the extracts were characterized using quantum chemical method; flavonoids and hydroxycinnamic acids were found to have similar redox parameters. Silver nanoparticles (AgNPs) colloids were synthesized using three Stevia extracts; AgNPs size distribution were characterized by means of scanning electron microscopy. All the extracts revealed significant activity in AgNPs synthesis; the nanoparticles of predominantly spherical shape with the average sizes of 16-25 nm were formed. The reducing properties of the extracts were found to correlate with total phenol content; the activity of extracts from the leaves of plants grown ex situ and from callus culture in Ag+ ions reduction was similar to each other and exceeded the activity of extract from the leaves of plants grown in vitro.

Exploring of bioactive compounds in essential oil acquired from the stem and root derivatives of Hypericum triquetrifolium callus cultures.

PubMed

Tahir, Nawroz Abdul-Razzak; Azeez, Hoshyar Abdullah; Muhammad, Kadhm Abdullah; Faqe, Shewa Anwer; Omer, Dlshad Ali

2017-12-25

The chemical profile of the essential oil of callus and cell suspension cultures derivatives from stem and root of Hypericum triquetrifolium were explored by ITEX/GC-MS. The major constituents for stem derivatives were undecane (78.44%) and 2,4,6-trimethyl-octane (9.74%) for fresh calli, 2,4-dimethyl-benzaldehyde (46.94%), 2,3-dimethyl-undecane (28.39%), 2,4-dimethyl-1-hexene (10.17%), 1,2-oxolinalool (3.64%) and limonene (3.55%) for dry calli and undecane (61.24%), octane, 2,4,6-trimethyl- (16.73%), nonane, 3-methyl-(3.74%), 2,5-diphenyl-benzoquinone (3.70%) and limonene (3.60%) for cell suspension. However, for root derivatives, the dominated components were: undecane (49.94%), eucalyptol (12.07%), limonene (9.98%), toluene (9.03%) and 3-methyl-nonane (4.29%) for fresh calli, 2,4-dimethyl-benzaldehyde (29.80%), 1,1-dimethylethyl-cyclohexane (14.99%), 3-methyl-pentanal (14.99%), undecane (10.04%), beta-terpinyl acetate (8.60%), 1,2-oxolinalool (6.27%) and 2-pentyl-furan (4.09%) for dry calli, undecane (52.38%), 2,4,6-trimethyl-octane (13.81%), 3-methyl-nonane (5.73%), toluene (4.82%) and limonene (4.57%) for cell suspension derivative in root. The attained outcomes indicated that the alkane, aldehyde and monoterpene fractions dominated the chemical composition of essential oils.

Zinc and copper tolerance of Agrostis stolonifera L. in tissue culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, L.; Antonovics, J.

1978-03-01

Callus tissue was induced from shoot meristematic tissue and root tips of a clone of the grass Agrostis stolonifera tolerant to both zinc and copper, and from a control clone tolerant to neither metal. Growth of the callus tissue on media containing zinc and copper showed that tolerance to both metals was maintained in tissue culture. The pattern of metal uptake in tissue culture resembled uptake by whole plants in that tolerant tissue took up more metal than nontolerant tissue. Plants regenerated from callus had the same copper and zinc tolerance as the original parental clones regardless of time ofmore » growth in tissue culture and shoot or root origin of the tissue. The results support previous evidence that metal tolerance is genetically determined and acts at the cellular level.« less

Micropropagation and organogenesis of Anthurium andreanum Lind cv Rubrun.

PubMed

Maira, Oropeza; Alexander, Mejías; Vargas, Teresa Edith

2010-01-01

Tissue culture techniques are routinely used for mass propagation and the establishment of disease free stock material. Virtually all pot type Anthuriums available in the market today are produced by tissue culture. In this chapter, we describe an efficient protocol to obtain Anthurium andreanum cv Rubrun vitro plants through micropropagation and organogenesis. Seeds from plant spadixes were germinated on MS medium supplemented with 0.5 mg/L BA. Micro-cuttings from in vitro germinated seedlings were subcultured on MS medium containing 2 mg/L BA and 0.5 mg/L NAA. Four-week-old in vitro plants obtained from microcuttings, showed callus proliferation at the stem base. The development of shoots and plantlets was observed from callus tissue. We also describe a detailed method for the histological analysis of callus tissue and a vitro plants acclimatization protocol.

Chemical characterization and anti-inflammatory effect of rauvolfian, a pectic polysaccharide of Rauvolfia callus.

PubMed

Popov, S V; Vinter, V G; Patova, O A; Markov, P A; Nikitina, I R; Ovodova, R G; Popova, G Yu; Shashkov, A S; Ovodov, Yu S

2007-07-01

The pectic polysaccharide named rauvolfian RS was obtained from the dried callus of Rauvolfia serpentina L. by extraction with 0.7% aqueous ammonium oxalate. Crude rauvolfian RS was purified using membrane ultrafiltration to yield the purified rauvolfian RSP in addition to glucan as admixture from the callus, with molecular weights 300 and 100-300 kD, respectively. A peroral pretreatment of mice with the crude and purified samples of rauvolfian (RS and RSP) was found to decrease colonic macroscopic scores, the total area of damage, and tissue myeloperoxidase activity in colons as compared with a colitis group. RS and RSP were shown to stimulate production of mucus by colons of the colitis mice. RSP appeared to be an active constituent of the parent RS. The glucan failed to possess anti-inflammatory activity.

Desferrioxamine for Stimulation of Fracture Healing and Revascularization in a Bone Defect Model

DTIC Science & Technology

2012-02-01

cartilaginous tissue still present. DBM + L-DFO: Fracture gap less evident with more complete bone bridging with denser trabecular bone and less...fracture callus volume by micro-CT, and qualitative histology for callus tissue quality and vascularity in 5 groups (No implant, CS implant, DFO+CS...Weinhold, P. North Carolina Tissue Engineering and Regenerative Medicine Meeting, November 4, 2011; Winston Salem, NC. (presented) • Desferroxamine with

Study of factors affecting growth and cold acclimation of Vitis callus cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, L.

1987-01-01

In vitro grape tissue culture initiation, growth, and cold acclimation were studied. Factors involved were genotypes, media, plant growth regulators, age, light, temperature, antioxidant, clearing and adsorbing agents, sucrose level, osmotic potential, ABA, chilling and freezing treatments. Murashige and Skoog (MS) medium containing 1 ..mu..M 2,4-d + 0.1 uM Ba, MS containing 1 uM 2,4-D, and woody plant medium containing 1 uM 2,4-D + 0.1 uM BA produced abundant callus tissue for most grape genotypes; either WPM or MS containing 1 uM BA stimulated shoot growth in all the 12 genotypes tested. Adding 1 uM abscisic acid (ABA) to themore » B5 medium with 1 uM 2,4-D and 0.5 uM BA enhanced growth and quality of Chancellor callus. /sup 3/H-ABA was taken up actively by callus tissue at 12 days after subculture, but by 20 d this effect disappeared. When /sup 14/C-sucrose was added to the medium. /sup 14/C level of cells reached a plateau after 48 h; this plateau was higher if ABA was also present in the medium. Cells on media containing ABA were larger in size, lighter in color, and more loosely connected.« less

Generation and multiplication of plantlets from callus derived from Haplopappus gracilus (Nutt.) Gray and their karyotype analysis

NASA Technical Reports Server (NTRS)

Kann, R. P.; O'Connor, S. A.; Levine, H. G.; Krikorian, A. D.

1991-01-01

Unopened flower heads of Haplopappus gracilis (2n = 4) provided primary explants for callus production and subsequent induction of organized growth. Callus was initiated from small (3-5 mm in length) floral buds with benzylaminopurine (BAP) (44.4 micromoles; 10 mg/l) and naphthalene acetic acid (NAA) (0.54 micromole; 0.1 mg/l). Lowering the BAP level to 4.44 micromoles (1 mg/l) but maintaining the NAA level, gave rise to organized but highly compressed shoot growing points from an otherwise undifferentiated callus mass. Shoots selected from such cultures were maintainable and could be proliferated by growing 1-1.5-cm stem tip cuttings on Murashige and Skoog basal medium (solidified with agar) containing 0.444 micromole (0.1 mg/l) BAP and 0.054 micromole (0.01 mg/l) NAA. The stem tip multiplication rates obtainable by these means permit reliable strategies for shoot multiplication or production of rooted plantlets. Prolonged subculture and maintenance of shoots on growth regulator-free medium leads to in vitro flowering and greatly reduces rooting capacity. Karyotype analysis of chromosomes from root tip cells at metaphase and chromosome measurements show that karyologically uniform plantlets (based on chromosome number and morphology) can be obtained.

Uptake, Distribution, and Metabolism of 1-Naphthaleneacetic Acid and Indole-3-Acetic Acid During Callus Initiation From Actinidia deliciosa Tissues.

PubMed

Centeno; Fernández; Feito; Rodríguez

1999-10-01

1-Naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) were required for in vitro callus formation at the basal edge of kiwifruit (Actinidia deliciosa [A. Chev] Liang and Ferguson, cv. Hayward) petioles. The uptake, metabolism, and concentration of NAA and indole-3-acetic acid (IAA) content were examined in the explants during the callus initiation period. After 1, 6, 12, 24, 48, and 96 h of culture in the presence of [H(3)]NAA, petioles were divided into apical, middle, and basal portions and analyzed. Except for a high IAA level measured at 12 h, IAA content decreased in tissues during a culture period of 96 h. NAA uptake was higher in petiolar edges than in the middle portion, and NAA was rapidly conjugated with sugars and aspartic acid inside the tissues. The amide conjugation was triggered in apical and basal portions from 12 h and in the middle part from 48 h, with alpha-naphthylacetylaspartic acid being the major metabolite. Free-NAA concentration in cultured petioles achieved an equilibrium with the exogenously applied NAA (0.27 µm) from 12 h, and it remained constant thereafter. The relationships between the role attributed to NAA and BA in the initiation and the maintenance of disorganized growth of callus in kiwifruit cultures are discussed.

Aseptic Culture Systems of Radopholus similis for In Vitro Assays on Musa spp. and Arabidopsis thaliana

PubMed Central

Elsen, A.; Lens, K.; Nguyet, D. T. M.; Broos, S.; Stoffelen, R.; De Waele, D.

2001-01-01

Radopholus similis is one of the most damaging nematodes in bananas. Chemical control is currently the most-used method, but nematode control through genetic improvement is widely encouraged. The objective of this study was to establish an aseptic culture system for R. similis and determine whether R. similis can infect and reproduce on in vitro banana plantlets and in vitro Arabidopsis thaliana. In the study's first part, a suitable aseptic culture system was developed using alfalfa callus. Radopholus similis could penetrate and reproduce in the callus. Six weeks after inoculation with 25 females, the reproduction ratio was 26.3 and all vermiform stages were present. The reproduction ratio increased to 223.2 after 12 weeks. Results of a greenhouse test showed that R. similis did not lose its pathogenicity after culturing on alfalfa callus. In the study's second part, the infection and reproduction of the nematodes cultured on the callus were studied on both in vitro banana plantlets and A. thaliana. Radopholus similis infected and reproduced on both banana and A. thaliana. Furthermore, nematode damage was observed in the root systems of both hosts. These successful infections open new perspectives for rapid in vitro screening for resistance in banana cultivars and anti-nematode proteins expressed in A. thaliana. PMID:19266012

Radiofrequency radiation effects on the common bean

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thomkins, K.; Griggs, L.; Myles, E.L.

Our environment is bombarded daily with thousands of objects we can visually detect. However, invisible to humans are the electromagnetic waves that penetrate our environment. Electromagnetic waves consist of a large spectrum of waves including the harmful gamma rays, x-rays, and ultraviolet rays. The question that has increased tremendously is: can low energy electromagnetic waves become harmful to living organisms? The purpose of this study is to determine the effect of radiofrequency radiation on protein synthesis of the common bean. Phaseolus vulgaris (kidney bean) was surface-sterilized and allowed to germinate on Mushurage and Skoog`s medium for 1 week. Hypocotyls weremore » wounded and placed on media to initiate callus production. Six petri dishes containing 1 g of callus were used in the experiment. Three dishes were exposed to 100kH in a Crawford cell for 24h. The remaining three petri dishes with callus were used as a control. After the exposure period, the protein from callus was extracted and analyzed by one-dimensional gel electrophoresis. The results show that hypocotyl growth was not different between control and experimental groups after 24 h. The result of one-dimensional gel electrophoresis did not show observable differences in protein synthesized by the control and experimental groups. Analysis of protein synthesis is still ongoing.« less

Embryogenesis induction, callogenesis, and plant regeneration by in vitro culture of tomato isolated microspores and whole anthers.

PubMed

Seguí-Simarro, José M; Nuez, Fernando

2007-01-01

In this work, some of the different in vitro developmental pathways into which tomato microspores or microsporocytes can be deviated experimentally were explored. The two principal ones are direct embryogenesis from isolated microspores and callus formation from meiocyte-containing anthers. By means of light and electron microscopy, the process of early embryogenesis from isolated microspores and the disruption of normal meiotic development and change of developmental fate towards callus proliferation, morphogenesis, and plant regeneration have been shown. From microspores isolated at the vacuolate stage, embryos can be directly induced, thus avoiding non-androgenic products. In contrast, several different morphogenic events can be triggered in cultures of microsporocyte-containing anthers under adequate conditions, including indirect embryogenesis, adventitious organogenesis, and plant regeneration. Both callus and regenerated plants may be haploid, diploid, and mostly mixoploid. The results demonstrate that both gametophytic and sporophytic calli occur in cultured tomato anthers, and point to an in vitro-induced disturbance of cytokinesis and subsequent fusion of daughter nuclei as a putative cause for mixoploidy and genome doubling during both tetrad compartmentalization and callus proliferation. The potential implications of the different alternative pathways are discussed in the context of their application to the production of doubled-haploid plants in tomato, which is still very poorly developed.

Characterization of a salt-responsive 24-kilodalton glycoprotein in Mesembryanthemum crystallinum.

PubMed Central

Yen, H E; Edwards, G E; Grimes, H D

1994-01-01

A concanavalin A (Con A)-binding polypeptide with a molecular mass of 24 kD (termed "SRgp24") was associated with the intercellular space of Mesembryanthemum crystallinum L. callus. When callus was grown in medium containing between 0 and 100 mM NaCl, SRgp24 was detected by Con A binding. Increasing the NaCl concentration to 200 mM caused a reduction in the amount of SRgp24 within 3 d, and returning the callus to medium without salt resulted in an accumulation of SRgp24. Immunoblot analysis showed that appreciable amounts of SRgp24 accumulated in the leaves when plants were grown under sodium-limiting conditions. Unlike most of the cell-wall Con A-binding proteins in M. crystallinum callus, the carbohydrate moiety of SRgp24 was resistant to endoglycosidase H digestion. After purification of SRgp24, the N terminus was sequenced and found to share 55 to 60% identity with the N terminus of osmotin, a group 5 pathogenesis-related protein (PR-5) that accumulates in salt-adapted tobacco cell suspension. Immunocytochemical assays, with affinity-purified antibodies to SRgp24, indicated that SRgp24 preferentially accumulated in the cell-wall region. We conclude that SRgp24 is a salt-responsive glycoprotein related to the PR-5 family in M. crystallinum. PMID:7972493

Production of gymnemic acid depends on medium, explants, PGRs, color lights, temperature, photoperiod, and sucrose sources in batch culture of Gymnema sylvestre.

PubMed

Ahmed, A Bakrudeen Ali; Rao, A S; Rao, M V; Taha, Rosna Mat

2012-01-01

Gymnema sylvestre (R.Br.) is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA). The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L) and KN (0.5 mg/L). Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w) was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w). Maximum GA production (58.28 mg/g d.w) was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors.

Spatially offset raman spectroscopy for non-invasive assessment of fracture healing

NASA Astrophysics Data System (ADS)

Ding, Hao; Lu, Guijin; West, Christopher; Gogola, Gloria; Kellam, James; Ambrose, Catherine; Bi, Xiaohong

2016-02-01

Fracture non-unions and bone re-fracture are common challenges for post-fracture management. To achieve better prognosis and treatment evaluation, it is important to be able to assess the quality of callus over the time course of healing. This study evaluated the potential of spatially offset Raman spectroscopy for assessing the fracture healing process in situ. We investigated a rat model of fracture healing at two weeks and 4 weeks post fracture with a fractured femur and a contralateral control in each animal. Raman spectra were collected from the depilated thighs on both sides transcutaneously in situ with various source/detection offsets. Bone signals were recovered from SORS spectra, and then compared with those collected from bare bones. The relative intensity of mineral from fractured bone was markedly decreased compared to the control. The fractured bones demonstrated lower mineral and carbonate level and higher collagen content in the callus at the early time point. Compared to week 2, collagen mineralization and mineral carbonation increased at 4 weeks post fracture. Similarly, the material properties of callus determined by reference point indentation also increased in the 4-week group, indicating improved callus quality with time. The results from Raman analysis are in agreement with radiographic and material testing, indicating the potential of this technique in assessing fracture healing in vivo.

Production and immunogenicity of Actinobacillus pleuropneumoniae ApxIIA protein in transgenic rice callus.

PubMed

Kim, Mi-Young; Kim, Tae-Geum; Yang, Moon-Sik

2017-04-01

Actinobacillus pleuropneumoniae is a major etiological agent that is responsible for swine pleuropneumonia, a highly contagious respiratory infection that causes severe economic losses in the swine production industry. ApxIIA is one of the virulence factors in A. pleuropneumoniae and has been considered as a candidate for developing a vaccine against the bacterial infection. A gene encoding an ApxIIA fragment (amino acids 439-801) was modified based on a plant-optimized codon and constructed into a plant expression vector under the control of a promoter and the 3' UTR of the rice amylase 3D gene. The plant expression vector was introduced into rice embryogenic callus (Oryza sativa L. cv. Dongjin) via particle bombardment-mediated transformation. The integration and transcription of the ApxIIA 439-801 gene were confirmed by using genomic DNA PCR amplification and Northern blot analysis, respectively. The synthesis of ApxIIA 439-801 antigen protein in transgenic rice callus was confirmed by western blot analysis. The concentration of antigen protein in lyophilized samples of transgenic rice callus was 250 μg/g. Immunizing mice with protein extracts from transgenic plants intranasally elicited secretory IgA. These results demonstrate the feasibility of using a transgenic plant to elicit immune responses against A. pleuropneumoniae. Copyright © 2017 Elsevier Inc. All rights reserved.

Nandrolone decanoate appears to increase bone callus formation in young adult rats after a complete femoral fracture.

PubMed

Guimarães, Ana Paula Franttini Garcia Moreno; Butezloff, Mariana Maloste; Zamarioli, Ariane; Issa, João Paulo Mardegan; Volpon, José Batista

2017-11-01

To evaluate the influence of nandrolone decanoate on fracture healing and bone quality in normal rats. Male rats were assigned to four groups (n=28/group): Control group consisting of animals without any intervention, Nandrolone decanoate (DN) group consisting of animals that received intramuscular injection of nandrolone decanoate, Fracture group consisting of animals with a fracture at the mid-diaphysis of the femur, and Fracture and nandrolone decanoate group consisting of animals with a femur fracture and treatment with nandrolone decanoate. Fractures were created at the mid-diaphysis of the right femur by a blunt trauma and internally fixed using an intramedullary steel wire. The DN was injected intramuscularly twice per week (10 mg/kg of body mass). The femurs were measured and evaluated by densitometry and mechanical resistance after animal euthanasia. The newly formed bone and collagen type I levels were quantified in the callus. The treated animals had longer femurs after 28 days. The quality of the intact bone was not significantly different between groups. The bone callus did show a larger mass in the treated rats. The administration of nandrolone decanoate did not affect the quality of the intact bone, but might have enhanced the bone callus formation.

Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

PubMed

Sahu, Jyoti; Khan, Shagufta; Sahu, Ram Kumar; Roy, Amit

2014-04-01

Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.

Plant germination and production of callus from the yellow hornpoppy (Glaucium flavum): the first stage of micropropagation.

PubMed

Mohamed, M E; Arafa, A M; Soliman, S S; Eldahmy, S I

2014-09-01

The yellow hornpoppy, Glaucium flavum Cr. (Fam. Papaveraceae) is a perennial herb, distributed in the Mediterranean region, including Egypt. The plant contains many benzyl isoquinoline alkaloids from the aporphine type such as glaucine, isoboldine, 1-chelidonine, 1-norchelidonine and 3-O-methylarterenol, making it to display various medicinal activities including antitussive, anticancer, antioxidant, antimicrobial, antiviral, hypoglycemic, analgesic, antipyretic, bronchodilator and anti-inflammatory effects. The plant is now rare and endangered in the Egyptian flora due to urban sprawl. The present study looks into Glaucium flavum seeds' in vitro germination as well as the ability of the explants taken from the growing seedlings to form stable callus lines in order to enable micropropagation as a way to save the rare plant. The study also scans the production of different medicinally valuable alkaloids, particularly glaucine, in produced callus.

Effect of medium modification and selected precursors on sterol production by short-term callus cultures of Euphorbia tirucalli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biesboer, D.D.; Mahlberg, P.G.

1979-01-01

Latex from E. Tirucalli, a potential rubber source, contains steroidal alcohols that are high in energy and thus of value in biomass conversion to fuels. Euphol was present in large amounts in the latex, but tirucallol predominated in greater quantities in explants and callus indicating synthesis and/or accumulation of tirucallol by cells other than the laticifer cell. Sterol production was significantly enhanced by certain nutrient media, as well as indole-3-acetic acid, and depressed by benzyladenine. Precursor stimulation of product synthesis was successful only with squalene, which promoted sterol production at 1.0 mg/liter but inhibited cell growth at higher concentrations. DL-mevalonicmore » acid and lanosterol promoted neither growth nor sterol production. DL-(214C) mevalonate was used to confirm the biosynthesis of sterols in both latex and callus cultures.« less

[Influence of genotype, explant type and component of culture medium on in vitro callus induction and shoot organogenesis of tomato (Solanum lycopersicum L.)].

PubMed

Khaliluev, M R; Bogoutdinova, L R; Baranova, G B; Baranova, E N; Kharchenko, P N; Dolgov, S V

2014-01-01

The influence of explant type as well as of the type of growth regulators and concentration on callus induction processes and somatic organogenesis of shoots was studied in vitro on four tomato genotypes of Russian breeding. Cytological study of callus tissue was conducted. It was established that tomato varieties possess a substantially greater ability to indirect shoot organogenesis compared with the F1 hybrid. The highest frequency of somatic organogenesis of shoots, as well as their number per explant, was observed for most of the genotypes studied during the cultivation of cotyledons on Murashige-Skoog culture medium containing 2 mg/l of zeatin in combination with 0.1 mg/l of 3-indoleacetic acid. An effective protocol of indirect somatic organogenesis of shoots from different explants of tomato varieties with a frequency of more than 80% was developed.

Production of haploids from anther culture of banana [Musa balbisiana (BB)].

PubMed

Assani, A; Bakry, F; Kerbellec, F; Haïcour, R; Wenzel, G; Foroughi-Wehr, B

2003-02-01

We report here, for the first time, the production of haploid plants of banana Musa balbisiana (BB). Callus was induced from anthers in which the majority of the microspores were at the uninucleate stage. The frequency of callus induction was 77%. Callus proliferation usually preceded embryo formation. About 8% of the anthers developed androgenic embryos. Of the 147 plantlets obtained, 41 were haploids (n=x=11). The frequency of haploid production depended on genotypes used: 18 haploid plants were produced from genotype Pisang klutuk, 12 from Pisang batu, seven from Pisang klutuk wulung and four from Tani. The frequency of regeneration was 1.1%, which was based on the total number of anthers cultured. Diploid plants (2n=2x=22) were also observed in the regenerated plants. The haploid banana plants that were developed will be important material for the improvement of banana through breeding programmes.

Human Bone-Forming Chondrocytes Cultured in the Hydrodynamic Focusing Bioreactor Retain Matrix Proteins: Similarities to Spaceflight Results

NASA Technical Reports Server (NTRS)

Duke, P. J.; Hecht, J.; Montufar-Solis, D.

2006-01-01

Fracture healing, crucial to a successful Mars mission, involves formation of a cartilaginous fracture callus which differentiates, mineralizes, ossifies and remodels via the endochondral process. Studies of spaceflown and tailsuspended rats found that, without loading, fracture callus formation and cartilage differentiation within the callus were minimal. We found delayed differentiation of chondrocytes within the rat growth plate on Cosmos 1887, 2044, and Spacelab 3. In the current study, differentiation of human bone-forming chondrocytes cultured in the hydrodynamic focusing bioreactor (HFB) was assessed. Human costochondral chondrocytes in suspension were aggregated overnight, then cultured in the HFB for 25 days. Collagen Type II, aggrecan and unsulfated chondroitin were found extracellularly and chondroitin sulfates 4 and 6 within the cell. Lack of secretion was also found in pancreatic cells of spaceflown rats, and in our SL3 studies. The HFB can be used to study cartilage differentiation in simulated microgravity.

Hypergravity Leads to the Redistribution of Calcium Ions in Plant Cell

NASA Astrophysics Data System (ADS)

Nedukha, Olena M.

2008-06-01

The study of hypergravity influence on calcium ions distribution and on the relative amount of Ca2+ in cells of Nicotiana tabacum callus was carried out using the centrifuge. 15-day-old N. tabacum callus grown in a Murashige and Scoog agar medium was exposed to hypergravity at 6.5 g and 14 g for 15 and 60 min. The control samples and the centrifuged callus were loaded with Fluo-4 and then studied by the confocal laser-scanning microscopy. The visible redistribution of Ca2+ in the investigated cells and the appearance of calcium-microdomains in cytoplasm have been established under influence of hypergravity. Readaptation of Ca2+ distribution in the cells occurred in 2-4 h after hypergravity ending. It is suggested that influence of hypergravity lead to change of ionic transport of plasmalemma and endomembranes, and also to efflux of Ca2+ from apoplast.

Chemical Compositions, Somatic Embryogenesis, and Somaclonal Variation in Cumin

PubMed Central

Tohidfar, Masoud; Sadat Noori, Seyed Ahmad; Izadi Darbandi, Ali; Rao, Rosa

2017-01-01

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis of the content of catalase, ascorbate peroxidase, proline, protein, terpenic compounds, alcohol/phenols, aldehydes, and epoxides) and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan). Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 0.1 mg/L kinetin proved to be the best primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet. Results indicated that different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potential. The high content of catalase, ascorbate peroxidase, proline, and terpenic hydrocarbons and low content of alcoholic and phenolic compositions had a stimulatory effect on somatic embryogenesis. Band patterns of RAPD markers in regenerated plants were different from those of the mother plants. This may be related to somaclonal variations or pollination system of cumin. Generally, measurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cumin. Also, somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs. PMID:29234682

In vitro antibacterial, antifungal, and antioxidant activities of the essential oil and methanol extracts of herbal parts and callus cultures of Satureja hortensis L.

PubMed

Güllüce, M; Sökmen, M; Daferera, D; Ağar, G; Ozkan, H; Kartal, N; Polissiou, M; Sökmen, A; Sahin, F

2003-07-02

The present study was designated to evaluate the antimicrobial and antioxidant activities of the essential oil, obtained by using a Clevenger distillation apparatus, water soluble (polar) and water insoluble (nonpolar) subfractions of the methanol extracts from aerial parts of Satureja hortensis L. plants, and methanol extract from calli established from the seeds using Gamborg's B5 basal media supplemented with indole-3-butyric acid (1.0 ppm), 6-benzylaminopurine (N(6)-benzyladenine) (1.0 ppm), and sucrose (2.5%). The antimicrobial test results showed that the essential oil of S. hortensis had great potential antimicrobial activities against all 23 bacteria and 15 fungi and yeast species tested. In contrast, the methanol extract from callus cultures and water soluble subfraction of the methanol extract did not show antimicrobial activities, but the nonpolar subfraction had antibacterial activity against only five out of 23 bacterial species, which were Bacillus subtilis, Enterococcus fecalis, Pseudomonas aeruginosa, Salmonella enteritidis, and Streptococcus pyogenes. Antioxidant studies suggested that the polar subfractions of the methanol extract of intact plant and methanol extract of callus cultures were able to reduce the stable free radical 2,2-diphenyl-1-picrylhydrazyl to the yellow-colored diphenylpicrylhydrazine. In this assay, the strongest effect was observed for the tissue culture extract, with an IC(50) value of 23.76 +/- 0.80 microgram/mL, which could be compared with the synthetic antioxidant agent butylated hydroxytoluene. On the other hand, linoleic acid oxidation was 95% inhibited in the presence of the essential oil while the inhibition was 90% with the chloroform subfraction of the intact plant. The chemical composition of a hydrodistilled essential oil of S. hortensis was analyzed by gas chromatography (GC)/flame ionization detection (FID) and a GC-mass spectrometry system. A total 22 constituents representing 99.9% of the essential oil were identified by GC-FID analaysis. Thymol (29.0%), carvacrol (26.5%), gamma-terpinene (22.6%), and p-cymene (9.3%) were the main components.

Effect of low-intensity pulsed ultrasound stimulation on gap healing in a rabbit osteotomy model evaluated by quantitative micro-computed tomography-based cross-sectional moment of inertia.

PubMed

Tobita, Kenji; Matsumoto, Takuya; Ohashi, Satoru; Bessho, Masahiko; Kaneko, Masako; Ohnishi, Isao

2012-07-01

It has been previously demonstrated that low-intensity pulsed ultrasound stimulation (LIPUS) enhances formation of the medullary canal and cortex in a gap-healing model of the tibia in rabbits, shortens the time required for remodeling, and enhances mineralization of the callus. In the current study, the mechanical integrity of these models was confirmed. In order to do this, the cross-sectional moment of inertia (CSMI) obtained from quantitative micro-computed tomography scans was calculated, and a comparison was made with a four-point bending test. This parameter can be analyzed in any direction, and three directions were selected in order to adopt an XYZ coordinate (X and Y for bending; Z for torsion). The present results demonstrated that LIPUS improved earlier restoration of bending stiffness at the healing site. In addition, LIPUS was effective not only in the ultrasound-irradiated plane, but also in the other two planes. CSMI may provide the structural as well as compositional determinants to assess fracture healing and would be very useful to replace the mechanical testing.

[CHANGES OF SEMAPHORIN 3A EXPRESSION IN HEALING OF TIBIA FRACTURE AFTER TRAUMATIC BRAIN INJURY].

PubMed

Li, Zhengzheng; Zhao, Junwei; Yi, Zhigang; Luo, Wei; Li, Kang; Wang, Yuliang; Wang, Jing; An, Liping; Ma, Jinglin

2016-10-08

To investigate the mechanism of Semaphorin 3A (Sema3A) in fracture healing after nerve injury by observing the expression of Sema3A in the tibia fracture healing after traumatic brain injury (TBI). A total of 192 Wistar female rats, 8-10 weeks old and weighing 220-250 g, were randomly divided into tibia fracture group (group A, n =48), TBI group (group B, n =48), TBI with tibia fracture group (group C, n =48), and control group (group D, n =48). The tibia fracture model was established at the right side of group A; TBI model was made in group B by the improved Feeney method; the TBI and tibia fracture model was made in group C; no treatment was given in group D. The tissue samples were respectively collected at 3, 5, 7, 14, 21, and 28 days after operation; HE staining, immunohistochemistry staining, and Western blot method were used for the location and quantitative detection of Sema3A in callus tissue. HE staining showed that no obvious changes were observed at each time point in groups B and D. At 3 and 5 days, there was no obvious callus growth at fracture site with inflammatory cells and fibrous tissue filling in groups A and C. At 7 and 14 days, fibrous tissue grew from periosteum to fracture site in groups A and C; the proliferation of chondrocytes in exterior periosteum gradually formed osteoid callus at fracture site in groups A and C. The chondrocyte had bigger size, looser arrangement, and more osteoid in group C than group A. Group B had disorder periosteum, slight subperiosteal bone hyperplasia, and no obvious change of bone trabecula in group B when compared with group D. At 21 and 28 days, cartilage callus was gradually replaced by new bone trabecula in groups A and C. Group C had loose arrange, disorder structure, and low density of bone trabecula, big callus area and few chondrocyte and osteoid when compared with group A; group B was similar to Group D. Immunohistochemistry staining showed that Sema3A expression in chondrocytes in group C was higher than that in group A, particularly at 7, 14, and 21 day. Sema3A was significantly higher in osteoblasts of new bone trabecula in group A than group C, especially at 14 and 21 days ( P <0.05). Western blot results showed that the Sema3A had the same expression trend during fracture healing in groups A and C. However, the expression of Sema3A protein was significantly higher in group C than group A ( P <0.05) and in group B than group D ( P <0.05) at 7, 14, 21, and 28 days. Abnormal expression of Sema3A may play a role in fracture healing after nerve injury by promoting the chondrocytes proliferation and reducing the distribution of sensory nerve fibers and osteoblast differentiation.

Short-Term Effects of Carbon Dioxide on Carnation Callus Cell Respiration 1

PubMed Central

Palet, Artur; Ribas-Carbó, Miquel; Argilés, Josep M.; Azcón-Bieto, Joaquim

1991-01-01

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O2 uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO2 in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO2 again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO2/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO2/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO2/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO2) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited. PMID:16668209

Effect of sucrose, erythrose-4-phosphate and phenylalanine on biomassa and flavonoid content of callus culture from leaves of Gynura procumbens Merr.

NASA Astrophysics Data System (ADS)

Nurisa, Aryana; Kristanti, Alfinda Novi; Manuhara, Yosephine Sri Wulan

2017-08-01

The aims of this study were to know the effect of concentration of sucrose, erythrose-4-phosphate and phenylalanine on biomass and flavonoid content of callus cultures from leaves of sambung nyawa (Gynura procumbens Merr.). This study was experimental research with complete randomized design. Callus induction was treated in MS medium supplemented with NAA 2 mg/L, BAP 1 mg/L and sucrose concentration (10 g/L, 30 g/L and 50 g/L) respectively were combined with erythrose-4-phosphate (0 µM, 2,5 µM and 5 µM) and phenylalanine (0 mg/L, 2 mg/L and 3 mg/L), each treatment were repeated four times. After six weeks of culture, fresh and dry weight of calli were measured and extracted with ethanol absolut. Crude extract ethanolic of callus was analyzed used by a modified colorimetric with spectrophotometer method. The best yield of calli biomass (0,672 ± 0,112 gram of fresh weight and 0,033 ± 0,009 gram of dry weight) was obtained in treatment of 30 g/L sucrose of and 5 µM erythrose-4-phosphate. The highest total flavonoid content was obtained of calli treated with 30 g/L of sucrose and 3 mg/L of phenylalanine (3633,4 ppm quercetin/gram dry weight and 15777,8 ppm kaempferol/gram dry weight).

Establishment and Phytochemical Analysis of a Callus Culture from Ageratina pichinchensis (Asteraceae) and Its Anti-Inflammatory Activity.

PubMed

Sánchez-Ramos, Mariana; Bahena, Silvia Marquina; Romero-Estrada, Antonio; Bernabé-Antonio, Antonio; Cruz-Sosa, Francisco; Gonzálesssz-Christen, Judith; Acevedo-Fernández, Juan José; Perea-Arango, Irene; Alvarez, Laura

2018-05-25

A protocol was established to produce bioactive compounds in a callus culture of Ageratina pichinchensis by using 1 mg L -1 NAA with 0.1 mg L -1 KIN. The phytochemical study of the EtOAc extract obtained from the callus biomass, allowed the isolation and characterization of eleven secondary metabolites, of which dihydrobenzofuran ( 5 ) and 3-epilupeol ( 7 ), showed important anti-inflammatory activity. Compound 5 inhibits in vitro the secretion of NO (IC 50 = 36.96 ± 1.06 μM), IL-6 (IC 50 = 73.71 ± 3.21 μM), and TNF-α (IC 50 = 73.20 ± 5.99 μM) in RAW (Murine macrophage cells) 264.7 macrophages, as well as the activation of NF-κB (40% at 150 μM) in RAW-blue macrophages, while compound 7 has been described that inhibit the in vivo TPA-induced ear edema, and the in vitro production of NO, and the PLA2 enzyme activity. In addition, quantitative GC-MS analysis showed that the anti-inflammatory metabolites 5 and 7 were not detected in the wild plant. Overall, our results indicated that A. pichinchensis can be used as an alternative biotechnological resource for obtaining anti-inflammatory compounds. This is the first report of the anti-inflammatory activity of compound 5 and its production in a callus culture of A. pichinchensis .

Production of Gymnemic Acid Depends on Medium, Explants, PGRs, Color Lights, Temperature, Photoperiod, and Sucrose Sources in Batch Culture of Gymnema sylvestre

PubMed Central

Ahmed, A. Bakrudeen Ali; Rao, A. S.; Rao, M. V.; Taha, Rosna Mat

2012-01-01

Gymnema sylvestre (R.Br.) is an important diabetic medicinal plant which yields pharmaceutically active compounds called gymnemic acid (GA). The present study describes callus induction and the subsequent batch culture optimization and GA quantification determined by linearity, precision, accuracy, and recovery. Best callus induction of GA was noticed in MS medium combined with 2,4-D (1.5 mg/L) and KN (0.5 mg/L). Evaluation and isolation of GA from the calluses derived from different plant parts, namely, leaf, stem and petioles have been done in the present case for the first time. Factors such as light, temperature, sucrose, and photoperiod were studied to observe their effect on GA production. Temperature conditions completely inhibited GA production. Out of the different sucrose concentrations tested, the highest yield (35.4 mg/g d.w) was found at 5% sucrose followed by 12 h photoperiod (26.86 mg/g d.w). Maximum GA production (58.28 mg/g d.w) was observed in blue light. The results showed that physical and chemical factors greatly influence the production of GA in callus cultures of G. sylvestre. The factors optimized for in vitro production of GA during the present study can successfully be employed for their large-scale production in bioreactors. PMID:22629221

Are bone turnover markers capable of predicting callus consolidation during bone healing?

PubMed

Klein, P; Bail, H J; Schell, H; Michel, R; Amthauer, H; Bragulla, H; Duda, G N

2004-07-01

The aim of this study was to determine the ability of the following bone turnover markers to monitor the course of callus consolidation during bone healing: Carboxy-terminal propeptide of procollagen type I (PICP), skeletal alkaline phosphatase (sALP), and amino-terminal propeptide of type III procollagen (PIlINP). Since interfragmentary movements have been proven to possess the ability to document the progression of bone healing in experimental studies, correlations between bone turnover markers and interfragmentary movements in vivo were investigated. Therefore, two different types of osteosyntheses representing different mechanical situations at the fracture site were compared in an ovine osteotomy model. Blood samples were taken preoperatively and postoperatively in weekly intervals over a nine-week healing period. At the same intervals, interfragmentary movements were measured in all sheep. After nine weeks, animals were sacrificed and the tibiae were evaluated both mechanically and histologically. Wide interindividual ranges were observed for all bone turnover markers. The systemic PICP level did not increase with callus consolidation. The bone-healing model seemed to influence the systemic level of PIIINP and sALP but no general correlation between bone turnover markers and interfragmentary movements could be detected. No differences between the different types of osteosyntheses and thus the different mechanical situations were observed. All analyzed markers failed as general predictors for the course of callus consolidation during bone healing.

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chien-Yuan; Donohoe, Bryon S.; Ahuja, Neha

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. After evaluatingmore » the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N 6 macronutrient/B 5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50-100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.« less

Piper nigrum: micropropagation, antioxidative enzyme activities, and chromatographic fingerprint analysis for quality control.

PubMed

Ahmad, Nisar; Abbasi, Bilal Haider; Rahman, Inayat ur; Fazal, Hina

2013-04-01

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg L(-1) 6-benzyladenine (BA) along with 0.5 mg L(-1) gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg L(-1) BA in combination with 0.25 mg L(-1) α-naphthalene acetic acid (NAA) and 0.25 mg L(-1) 2,4-dichlorophenoxyacetic acid or 0.5 mg L(-1) indole butyric acid (IBA) along with 0.25 mg L(-1) NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg L(-1) thiodiazoran or 1.5 mg L(-1) IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle "piperine." The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

PubMed

Ahmad, Nisar; Abbasi, Bilal Haider; Fazal, Hina; Khan, Mubarak Ali; Afridi, Muhammad Siddique

2014-01-01

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (<90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced>80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (<90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²⁺ chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications. Copyright © 2013 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum)

DOE PAGES

Lin, Chien-Yuan; Donohoe, Bryon S.; Ahuja, Neha; ...

2017-12-19

Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. After evaluatingmore » the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N 6 macronutrient/B 5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50-100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical β-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.« less

Piper sarmentosum enhances fracture healing in ovariectomized osteoporotic rats: a radiological study.

PubMed

Estai, Mohamed Abdalla; Suhaimi, Farihah Haji; Das, Srijit; Fadzilah, Fazalina Mohd; Alhabshi, Sharifah Majedah Idrus; Shuid, Ahmad Nazrun; Soelaiman, Ima-Nirwana

2011-01-01

Osteoporotic fractures are common during osteoporotic states. Piper sarmentosum extract is known to possess antioxidant and anti-inflammatory properties. To observe the radiological changes in fracture calluses following administration of a Piper sarmentosum extract during an estrogen-deficient state. A total of 24 female Sprague-Dawley rats (200-250 g) were randomly divided into 4 groups: (i) the sham-operated group; (ii) the ovariectomized-control group; (iii) the ovariectomized + estrogen-replacement therapy (ovariectomized-control + estrogen replacement therapy) group, which was supplemented with estrogen (100 μg/kg/day); and (iv) the ovariectomized + Piper sarmentosum (ovariectomized + Piper sarmentosum) group, which was supplemented with a water-based Piper sarmentosum extract (125 mg/kg). Six weeks after an ovariectomy, the right femora were fractured at the mid-diaphysis, and a K-wire was inserted. Each group of rats received their respective treatment for 6 weeks. Following sacrifice, the right femora were subjected to radiological assessment. The mean axial callus volume was significantly higher in the ovariectomized-control group (68.2 ± 11.74 mm³) than in the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups (20.4 ± 4.05, 22.4 ± 4.14 and 17.5 ± 3.68 mm³, respectively). The median callus scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups had median (range, minimum - maximum value) as 1.0 (0 - 2), 1.0 (1 - 2) and 1.0 (1 - 2), respectively, which were significantly lower than the ovariectomized-control group score of 2.0 (2 - 3). The median fracture scores for the sham-operated, estrogen-replacement-therapy and Piper sarmentosum groups were 3.0 (3 - 4), 3.0 (2 - 3) and 3.0 (2 - 3), respectively, which were significantly higher than the ovariectomized-control group score of 2.0 (1 - 2) (p<0.05). The Piper sarmentosum extract improved fracture healing, as assessed by the reduced callus volumes and reduced callus scores. This extract is beneficial for fractures in osteoporotic states.

Dynamic locked plating for fixation of distal femur fractures using near- cortical over-drilling: Preliminary results of a prospective observational study.

PubMed

Galal, Sherif

2017-01-01

Nonunion after locked plating of distal femur fractures is not uncommon. Authors wanted to assess if "Dynamic" locked plating using near-cortex over-Drilling technique would provide a mechanical environment the promotes callus formation, thereby avoiding non-union encountered when applying locked plates with the conventional method. This study was conducted at an academic Level 1 Trauma Center. This is a prospective study conducted from November 2015 to November 2016. Follow-up was 10 months on average (ranging from 8 to 12 months). The study included 20 patients with 20 fractures (13 males, 7 females). The average patients' age was 41.2 years (18-64 years). According to the Müller AO classification of distal femur fractures (33A-C) there were 15 cases with extra-articular fractures (AO 33A), 5 patients with intra-articular fractures (AO 33C). Dynamic Locked plating using near-cortical over-drilling technique was done for all patients. Two blinded observers assessed callus score on 6-week radiographs using a 4-point ordinal scale. A 2-tailed t -test. Two-way mixed intra-class correlation testing was performed to determine reliability of the callus measurements by the 2 observers. All patients achieved union, time to union was 13.4 weeks on average (range form 8-24 weeks). Delayed union was observed in 2 patients. The average callus score for fractures was 1.8 (SD 0.6). All fractures united in alignment except 1 fracture which united in valgus malalignment, the deformity was appreciated in the postoperative radiographs. No wound related complications, no loss of reduction, no catastrophic implant failure or screw breakage were detected. Dynamic locked plating using near-cortex over-drilling is a simple technique that uses standard locked plates that promotes callus formation when used for fixing distal femur fractures.

Thidiazuron Triggers Morphogenesis in Rosa canina L. Protocorm-Like Bodies by Changing Incipient Cell Fate.

PubMed

Kou, Yaping; Yuan, Cunquan; Zhao, Qingcui; Liu, Guoqin; Nie, Jing; Ma, Zhimin; Cheng, Chenxia; Teixeira da Silva, Jaime A; Zhao, Liangjun

2016-01-01

Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-ylurea; TDZ) is an artificial plant growth regulator that is widely used in plant tissue culture. Protocorm-like bodies (PLBs) induced by TDZ serve as an efficient and rapid in vitro regeneration system in Rosa species. Despite this, the mechanism of PLB induction remains relatively unclear. TDZ, which can affect the level of endogenous auxins and cytokinins, converts the cell fate of rhizoid tips and triggers PLB formation and plantlet regeneration in Rosa canina L. In callus-rhizoids, which are rhizoids that co-develop from callus, auxin and a Z-type cytokinin accumulated after applying TDZ, and transcription of the auxin transporter gene RcPIN1 was repressed. The expression of RcARF4, RcRR1, RcCKX2, RcCKX3, and RcLOG1 increased in callus-rhizoids and rhizoid tips while the transcription of an auxin response factor (RcARF1) and auxin transport proteins (RcPIN2, RcPIN3) decreased in callus-rhizoids but increased in rhizoid tips. In situ hybridization of rhizoids showed that RcWUS and RcSERK1 were highly expressed in columella cells and root stem cells resulting in the conversion of cell fate into shoot apical meristems or embryogenic callus. In addition, transgenic XVE::RcWUS lines showed repressed RcWUS overexpression while RcWUS had no effect on PLB morphogenesis. Furthermore, higher expression of the root stem cell marker RcWOX5 and root stem cell maintenance regulator genes RcPLT1 and RcPLT2 indicated the presence of a dedifferentiation developmental pathway in the stem cell niche of rhizoids. Viewed together, our results indicate that different cells in rhizoid tips acquired regeneration competence after induction by TDZ. A novel developmental pathway containing different cell types during PLB formation was identified by analyzing the endogenous auxin and cytokinin content. This study also provides a deeper understanding of the mechanisms underlying in vitro regeneration in Rosa.

Nonsterol Triterpenoids as Major Constituents of Olea europaea

PubMed Central

Stiti, Naïm; Hartmann, Marie-Andrée

2012-01-01

Plant triterpenoids represent a large and structurally diverse class of natural products. A growing interest has been focused on triterpenoids over the past decade due to their beneficial effects on human health. We show here that these bioactive compounds are major constituents of several aerial parts (floral bud, leaf bud, stem, and leaf) of olive tree, a crop exploited so far almost exclusively for its fruit and oil. O. europaea callus cultures were analyzed as well. Twenty sterols and twenty-nine nonsteroidal tetra- and pentacyclic triterpenoids belonging to seven types of carbon skeletons (oleanane, ursane, lupane, taraxerane, taraxastane, euphane, and lanostane) were identified and quantified by GC and GC-MS as free and esterified compounds. The oleanane-type compounds, oleanolic acid and maslinic acid, were largely predominant in all the organs tested, whereas they are practically absent in olive oil. In floral buds, they represented as much as 2.7% of dry matter. In callus cultures, lanostane-type compounds were the most abundant triterpenoids. In all the tissues analyzed, free and esterified triterpene alcohols exhibited different distribution patterns of their carbon skeletons. Taken together, these data provide new insights into largely unknown triterpene secondary metabolism of Olea europaea. PMID:22523691

Pepper, chili (Capsicum annuum).

PubMed

Min, Jung; Shin, Sun Hee; Jeon, En Mi; Park, Jung Mi; Hyun, Ji Young; Harn, Chee Hark

2015-01-01

Pepper is a recalcitrant plant for Agrobacterium-mediated genetic transformation. Several obstacles to genetic transformation remain such as extremely low transformation rates; the choice of correct genotype is critical; and there is a high frequency of false positives due to direct shoot formation. Here, we report a useful protocol with a suitable selection method. The most important aspect of the pepper transformation protocol is selecting shoots growing from the callus, which is referred to as callus-mediated shoot formation. This protocol is a reproducible and reliable system for pepper transformation.

Crown gall transformation of tobacco callus cells by cocultivation with Agrobacterium tumefaciens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muller, A.; Manzara, T.; Lurquin, P.F.

1984-09-17

Incubation of cells from squashed tobacco callus tissue with virulent Agrobacterium tumefaciens leads to the production of cells displaying a crown gall phenotype. In vitro crown gall transformation of dicotyledonous plant cells has been demonstrated after cocultivation of cell-wall regenerating mesophyll protoplasts with Agrobacterium tumefaciens cells. In addition, it has been shown that protoplasts freshly isolated from suspension cultures, when treated with A. tumefaciens spheroplasts and a fusogen, also generated cells displaying a typical crown gall phenotype, i.e., phytohormone-independent growth and opine synthesis. Subsequently, both techniques were used to transfer and express foreign genes in plant cells via A. tumefaciensmore » T-DNA integration. For practical purposes, it would be advantageous to be able to perform crown gall transformation of plant cells in tissue culture. The authors report here for the first time the production of Nicotiana tabacum crown gall cells after cocultivation of callus tissue with A. tumefaciens A136 cells. 11 references, 1 figure, 1 table.« less

Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss.

PubMed

Weld, R; Heinemann, J; Eady, C

2001-03-01

The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens. Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation. The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression. Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture. Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity. The remaining 7 cell lines contained no gfp sequences. Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced. In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hahne, G.; Hoffmann, F.

A serious problem in the technology of plant cell culture is that isolated protoplasts from many species are reluctant to divide. We have succeeded in inducing consecutive divisions in a naturally arrested system i.e., protoplasts from a hibiscus cell line, which do not divide under standard conditions and in an artificially arrested system i.e., colchicine-inhibited callus protoplasts of Nicotiana glutinosa, which do readily divide in the absence of colchicine. In both cases, the reinstallation of a net of cortical microtubules, which had been affected either by colchicine or by the protoplast isolation procedure, resulted in continuous divisions of the formerlymore » arrested protoplasts. Several compounds known to support microtubule assembly in vitro were tested for their ability to promote microtubule assembly in vivo. Best results were obtained by addition of dimethyl sulfoxide to the culture medium. Unlimited amounts of callus could be produced with the dimethyl sulfoxide method from protoplasts which never developed a single callus in control experiments. 30 references, 3 figures.« less

Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene.

PubMed

Li, D D; Shi, W; Deng, X X

2003-12-01

Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens. Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS). The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium. Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C. Almost no resistant callus was obtained if the cocultivation medium lacked AS. The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting. Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.

PubMed

Teixeira da Silva, Jaime A; Jha, Sumita

2016-11-01

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

Influence of organic supplements on production of shoot and callus biomass and accumulation of bacoside in Bacopa monniera (L.) Pennell.

PubMed

Parale, Anuradha; Barmukh, Rajkumar; Nikam, Tukaram

2010-04-01

Production of valuable secondary metabolites through plant cell or organ culture is the best suited alternative to extraction of whole plant material and to increase production of secondary metabolites in in-vitro systems, feeding precursor or intermediate metabolites is an obvious and popular approach. The present investigation was aimed to study the influence of feeding of organic supplements, glycine (0-125 μM), ferulic acid (0-200 μM), phenylalanine (0-200 μM), α-ketoglutaric acid (0-200 μM) and pyruvic acid (0-200 μM) on production of bacoside-A (a triterpenoid type secondary metabolite responsible for cognition effects) in shoot and callus biomass of Bacopa monniera (L.) Pennell. The shoots were raised in liquid Murashige and Skoog's (MS) medium fortified with 5 μM 6-benzyladenine (BA) and callus biomass on agar solidified MS medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4 -D) in conjunction with 5 μM 1-napthaleneacetic acid (NAA). Among the organic supplements used, 100 μM pyruvic acid effectively enhanced the production of bacoside-A in shoot as well as callus biomass. The bacoside-A content in in-vitro raised shoot biomass was 4.0 and 1.2 times higher as compared to control and shoot biomass of naturally grown plants respectively. Inclusion of pyruvic acid in MS medium for in-vitro shoot cultures of B. monniera, can be adapted for enhanced production of bacoside-A.

Short-term effects of carbon dioxide on carnation callus cell respiration. [Dianthus Caryophyllus L. ; Elodea canadensis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palet, A.; Ribas-Carbo, M.; Argiles, J.M.

1991-06-01

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O{sub 2} uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Minchx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO{sub 2} in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO{sub 2} again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO{sub 2}/bicarbonatemore » dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO{sub 2}/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO{sub 2}/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO{sub 2}) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited.« less

Establishment of the regeneration system for Vicia faba L.

PubMed

Bahgat, Shimaa; Shabban, Omer A; El-Shihy, Osama; Lightfoot, David A; El-Shemy, Hany A

2009-01-01

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba. Two Egyptian faba bean cultivars 'Giza 2' and '24 Hyto' were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5 mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean.

Light-induced biochemical variations in secondary metabolite production and antioxidant activity in callus cultures of Stevia rebaudiana (Bert).

PubMed

Ahmad, Naveed; Rab, Abdur; Ahmad, Nisar

2016-01-01

Stevia rebaudiana (S. rebaudiana) is a very important species with worldwide medicinal and commercial uses. Light is one of the major elicitors that fluctuate morphogenic potential and biochemical responses. In the present study, we investigated the effect of various spectral lights on biomass accumulation and secondary metabolite production in callus cultures of S. rebaudiana. Leaf explants were placed on Murashige and Skoog (MS) medium and exposed to various spectral lights. 6-Benzyle adenine (BA) and 2, 4-dichlorophenoxy acetic acid (2, 4-D; 2.0 mgl(-1)) were used for callus induction. The control light (16/8h) produced optimum callogenic response (92.73%) than other colored lights. Compared to other colored lights, control grown cultures displayed maximum biomass accumulation (5.78 gl(-1)) during a prolonged log phase at the 18th day of growth kinetics. Cultures grown under blue light enhanced total phenolic content (TPC; 102.32 μg/g DW), total flavonoid content (TFC; 22.07 μg/g DW) and total antioxidant capacity (TAC; 11.63 μg/g DW). On the contrary, green and red lights improved reducing power assay (RPA; 0.71Fe(II)g(-1) DW) and DPPH-radical scavenging activity (DRSA; 80%). Herein, we concluded that the utilization of colored lights is a promising strategy for enhanced production of antioxidant secondary metabolites in callus cultures of S. rebaudiana. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of field-grown genetically modified Zoysia grass on bacterial community structure.

PubMed

Lee, Yong-Eok; Yang, Sang-Hwan; Bae, Tae-Woong; Kang, Hong-Gyu; Lim, Pyung-Ok; Lee, Hyo-Yeon

2011-04-01

Herbicide-tolerant Zoysia grass has been previously developed through Agrobacterium-mediated transformation. We investigated the effects of genetically modified (GM) Zoysia grass and the associated herbicide application on bacterial community structure by using culture-independent approaches. To assess the possible horizontal gene transfer (HGT) of transgenic DNA to soil microorganisms, total soil DNAs were amplified by PCR with two primer sets for the bar and hpt genes, which were introduced into the GM Zoysia grass by a callus-type transformation. The transgenic genes were not detected from the total genomic DNAs extracted from 1.5 g of each rhizosphere soils of GM and non-GM Zoysia grasses. The structures and diversities of the bacterial communities in rhizosphere soils of GM and non-GM Zoysia grasses were investigated by constructing 16S rDNA clone libraries. Classifier, provided in the RDP II, assigned 100 clones in the 16S rRNA gene sequences library into 11 bacterial phyla. The most abundant phyla in both clone libraries were Acidobacteria and Proteobacteria. The bacterial diversity of the GM clone library was lower than that of the non- GM library. The former contained four phyla, whereas the latter had seven phyla. Phylogenetic trees were constructed to confirm these results. Phylogenetic analyses of the two clone libraries revealed considerable difference from each other. The significance of difference between clone libraries was examined with LIBSHUFF statistics. LIBSHUFF analysis revealed that the two clone libraries differed significantly (P〈0.025), suggesting alterations in the composition of the microbial community associated with GM Zoysia grass.

[Callus distraction].

PubMed

Giebel, G

1995-09-01

Fibroblast networks that form collagen and connect the two ends of bone develop in the haematoma after corticotomy. This regenerative tissue is vascularized and distracted. Even during the lengthening, mineralization starts. This starts at the ends created by the osteotomy, in the form of conical bony columns 200 microns thick, which grows towards each other in a manner reminiscent of stalagmites and stalactites, until the central, fibrous inner zone (growth zone) formed during distraction is completely mineralized. Connective tissue and bony bars are arranged lengthwise. As a rule, intramembranous callus formation takes place during distraction osteogenesis with no intermediate cartilaginous step.

Assessment of carbon fibre composite fracture fixation plate using finite element analysis.

PubMed

Saidpour, Seyed H

2006-07-01

In the internal fixation of fractured bone by means of bone-plates fastened to the bone on its tensile surface, an on-going concern has been the excessive stress shielding of the bone by the excessively-stiff stainless-steel plate. The compressive stress shielding at the fracture-interface immediately after fracture-fixation delays callus formation and bone healing. Likewise, the tensile stress shielding in the layer of bone underneath the plate can cause osteoporosis and decrease in tensile strength of this layer. In this study a novel forearm internal fracture fixation plate made from short carbon fibre reinforced plastic (CFRP) was used in an attempt to address the problem. Accordingly, it has been possible to analyse the stress distribution in the composite plates using finite-element modelling. A three-dimensional, quarter-symmetric finite element model was generated for the plate system. The stress state in the underlying bone was examined for several loading conditions. Based on the analytical results the composite plate system is likely to reduce stress-shielding effects at the fracture site when subjected to bending and torsional loads. The design of the plate was further optimised by reducing the width around the innermost holes.

Thidiazuron Triggers Morphogenesis in Rosa canina L. Protocorm-Like Bodies by Changing Incipient Cell Fate

PubMed Central

Kou, Yaping; Yuan, Cunquan; Zhao, Qingcui; Liu, Guoqin; Nie, Jing; Ma, Zhimin; Cheng, Chenxia; Teixeira da Silva, Jaime A.; Zhao, Liangjun

2016-01-01

Thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) is an artificial plant growth regulator that is widely used in plant tissue culture. Protocorm-like bodies (PLBs) induced by TDZ serve as an efficient and rapid in vitro regeneration system in Rosa species. Despite this, the mechanism of PLB induction remains relatively unclear. TDZ, which can affect the level of endogenous auxins and cytokinins, converts the cell fate of rhizoid tips and triggers PLB formation and plantlet regeneration in Rosa canina L. In callus-rhizoids, which are rhizoids that co-develop from callus, auxin and a Z-type cytokinin accumulated after applying TDZ, and transcription of the auxin transporter gene RcPIN1 was repressed. The expression of RcARF4, RcRR1, RcCKX2, RcCKX3, and RcLOG1 increased in callus-rhizoids and rhizoid tips while the transcription of an auxin response factor (RcARF1) and auxin transport proteins (RcPIN2, RcPIN3) decreased in callus-rhizoids but increased in rhizoid tips. In situ hybridization of rhizoids showed that RcWUS and RcSERK1 were highly expressed in columella cells and root stem cells resulting in the conversion of cell fate into shoot apical meristems or embryogenic callus. In addition, transgenic XVE::RcWUS lines showed repressed RcWUS overexpression while RcWUS had no effect on PLB morphogenesis. Furthermore, higher expression of the root stem cell marker RcWOX5 and root stem cell maintenance regulator genes RcPLT1 and RcPLT2 indicated the presence of a dedifferentiation developmental pathway in the stem cell niche of rhizoids. Viewed together, our results indicate that different cells in rhizoid tips acquired regeneration competence after induction by TDZ. A novel developmental pathway containing different cell types during PLB formation was identified by analyzing the endogenous auxin and cytokinin content. This study also provides a deeper understanding of the mechanisms underlying in vitro regeneration in Rosa. PMID:27200031

Enhanced production of phenolic acids in cell suspension culture of Salvia leriifolia Benth. using growth regulators and sucrose.

PubMed

Modarres, Masoomeh; Esmaeilzadeh Bahabadi, Sedigheh; Taghavizadeh Yazdi, Mohammad Ehsan

2018-04-01

Salvia leriifolia Benth. (Lamiaceae) is an endangered medicinal plant with hypoglycemic, anti-inflammatory and analgesic properties. Many of the beneficial effects of Salvia spp. are attributed to the phenolic compounds. In the present study, an efficient procedure has been developed for establishment of cell suspension culture of S. leriifolia as a strategy to obtain an in vitro phenolic acids producing cell line for the first time. The effect of growth regulators and various concentrations of sucrose have been analyzed, to optimize biomass growth and phenolic acids production. The callus used for this purpose was obtained from leaves of 15-day-old in vitro seedlings, on Murashige and Skoog (MS) basal medium supplemented with different hormone balances including benzylaminopurine (BAP) and indole butyric acid (IBA); 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN); naphthaleneacetic acid (NAA) and BAP. Modified MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA was the optimal condition for callus formation with the highest induction rate (100%), the best callus growth and the highest phenolic acids content. No callus induction was observed in combinations of IBA and BAP. Cell suspension cultures were established by transferring 0.5 g of callus to 30 mL liquid MS medium supplemented with 5 mg/L BAP and 5 mg/L NAA. Dynamics of phenolic acids production has been investigated during the growth cycle of the suspension cultures. The maximum content of caffeic acid and salvianolic acid B were observed on the 15th day of the cultivation cycle while the highest amount of rosmarinic acid was observed on the first day. In response to various sucrose concentrations, cell cultures with 40 g/L sucrose not only produced the highest dry biomass but also the highest induction of caffeic acid and salvianolic acid B. The highest amount of rosmarinic acid was observed in media containing 50 g/L sucrose. These prepared cell suspension cultures provided a useful system for further enhanced production of phenolic acids at a large scale.

Pharmacologically targeting beta-catenin for NF1 associated deficiencies in fracture repair.

PubMed

Baht, Gurpreet S; Nadesan, Puviindran; Silkstone, David; Alman, Benjamin A

2017-05-01

Patients with Neurofibromatosis type 1 display delayed fracture healing and the increased deposition of fibrous tissue at the fracture site. Severe cases can lead to non-union and even congenital pseudarthrosis. Neurofibromatosis type 1 is caused by a mutation in the NF1 gene and mice lacking the Nf1 gene show a fracture repair phenotype similar to that seen in patients. Tissue from the fracture site of patients with Neurofibromatosis type 1 and from mice deficient in the Nf1 gene both show elevated levels of β-catenin protein and activation of β-catenin mediated signaling. Constitutively elevated β-catenin leads to a delayed and fibrous fracture repair process, and (RS)-5-methyl-1-phenyl-1,3,4,6-tetrahydro-2,5-benzoxazocine (Nefopam, a centrally-acting, non-narcotic analgesic agent) inhibits β-catenin mediated signaling during skin wound repair. Here we investigate Nefopam's potential as a modulator of bone repair in mice deficient in Nf1. Mice were treated with Nefopam and investigated for bone fracture repair. Bone marrow stromal cells flushed from the long bones of unfractured mice were treated with Nefopam and investigated for osteogenic potential. Treatment with Nefopam was able to lower the β-catenin level and the Axin2 transcript level in the fracture calluses of Nf1 deficient mice. Cultures from the bone marrow of Nf1 -/- mice had significantly lower osteoblastic colonies and mineralized nodules, which was increased when cells were cultured in the presence of Nefopam. Fracture calluses were harvested and analyzed 14days and 21days after injury. Nf1 -/- calluses had less bone, less cartilage, and higher fibrous tissue content than control calluses. Treatment with Nefopam increased the bone and cartilage content and decreased the fibrous tissue content in Nf1 -/- calluses. These findings present a potential treatment for patients with Neurofibromatosis 1 in the context of bone repair. Since Nefopam is already in use in patient care, it could be rapidly translated to the clinical setting. Copyright © 2017 Elsevier Inc. All rights reserved.

Do estrogen and alendronate improve metaphyseal fracture healing when applied as osteoporosis prophylaxis?

PubMed

Kolios, Leila; Hoerster, Ann Kristin; Sehmisch, Stephan; Malcherek, Marie Christin; Rack, Thomas; Tezval, Mohammed; Seidlova-Wuttke, Dana; Wuttke, Wolfgang; Stuermer, Klaus Michael; Stuermer, Ewa Klara

2010-01-01

Osteoporosis is accompanied by predominantly metaphyseal fractures with a delayed and qualitatively reduced healing process. This study addressed the question of whether fracture healing in the context of osteoporosis prophylaxis is improved with estrogen (E) or alendronate (ALN). Thirty-six ovariectomized and 12 sham-operated 12-week-old rats received soy-free (osteoporotic C, sham), E-, or ALN- supplemented diets. After 10 weeks, a metaphyseal tibia osteotomy and standardized T-plate fixation were performed. After a 5-week healing process, the fracture callus was evaluated qualitatively by biomechanical bending test and quantitatively in microradiographic sections. The time course of callus formation was examined using fluorochrome-labeled histological sections. Administration of E improved the biomechanical properties of callus (stiffness [N/mm]: sham: 110.2 + or - 76.07, C: 41.28 + or - 33.70, E: 85.72 + or - 47.24, ALN: 72.07 + or - 34.68). The resistance to microfracturing seen in E-treated animals was significantly enhanced and even superior to sham (yield load [N] sham: 27.44 + or - 9.72, C: 21.04 + or - 12.47, E: 42.85 + or - 13.74(Delta), ALN: 25.28 + or - 6.4(.)) (* P < 0.05 vs. sham group, (Delta) P < 0.05 vs. C group, (*) P < 0.05 vs. E group). Trabecular bone in particular was improved, indicating the presence of physiological endosteal bridging (Tr.Dn [%] sham: 10.53 + or - 18.9, C: 1.01 + or - 0.14, E: 24.13 + or - 34.09(Delta), ALN: 3.99 + or - 8.3(.)). ALN did not help bone healing, as shown by mechanical tests. Compared to the C group, statistically, ALN did not show worse properties. The induction of callus formation under ALN treatment was slightly delayed (Tt.Cl [mm(2)] sham: 3.68 + or - 0.66, C: 3.44 + or - 0.42, E: 3.69 + or - 0.58, ALN: 3.06 + or - 0.56). Osteoporotic metaphyseal fracture healing was qualitatively and quantitatively improved by E prophylaxis. The process of fracture healing occurred nearly physiologically (shamlike). Notably, ALN hardly improved metaphyseal callus properties when assessed as osteoporosis prophylaxis, but to a lesser extent than E.

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.).

PubMed

El Abidine Triqui, Zine; Guédira, Abdelkarim; Chlyah, Averil; Chlyah, Hassane; Souvannavong, Vongthip; Haïcour, Robert; Sihachakr, Darasinh

2008-03-01

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 microM 2,4-D and 1 microM Kinetin or 5 microM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 microM 2,4,5-T or the combination of 10 microM 2,4-D and 1 microM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry.

Proliferation and ajmalicine biosynthesis of Catharanthus roseus (L). G. Don adventitious roots in self-built temporary immersion system

NASA Astrophysics Data System (ADS)

Phuc, Vo Thanh; Trung, Nguyen Minh; Thien, Huynh Tri; Tien, Le Thi Thuy

2017-09-01

Periwinkle (Catharanthus roseus (L.) G. Don) is a medicinal plant containing about 130 types of alkaloids that have important pharmacological effects. Ajmalicine in periwinkle root is an antihypertensive drug used in treatment of high blood pressure. Adventitious roots obtained from periwinkle leaves of in vitro shoots grew well in quarter-strength MS medium supplemented with 0.3 mg/l IBA and 20 g/l sucrose. Dark condition was more suitable for root growth than light. However, callus formation also took place in addition to the growth of adventitious roots. Temporary immersion system was applied in the culture of adventitious roots in order to reduce the callus growth rate formed in shake flask cultures. The highest growth index of roots was achieved using the system with 5-min immersion every 45 min (1.676 ± 0.041). The roots cultured in this system grew well without callus formation. Ajmalicine content was highest in the roots cultured with 5-min immersion every 180 min (950 μg/g dry weight).

The effects of axial displacement on fracture callus morphology and MSC homing depend on the timing of application.

PubMed

Weaver, Aaron S; Su, Yu-Ping; Begun, Dana L; Miller, Joshua D; Alford, Andrea I; Goldstein, Steven A

2010-07-01

The local mechanical environment and the availability of mesenchymal stem cells (MSC) have both been shown to be important factors in bone fracture healing. This study was designed to investigate how the timing of an applied axial displacement across a healing fracture affects callus properties as well as the migration of systemically introduced MSC. Bilateral osteotomies were created in male, Sprague-Dawley rats. Exogenous MSC were injected via the tail vein, and a controlled micro-motion was applied to one defect starting 0, 3, 10, or 24 days after surgery. The results showed that fractures stimulated 10 days after surgery had more mineral, less cartilage, and greater mechanical properties at 48 days than other groups. Populations of MSC were found in osteotomies 48 days after surgery, with the exception of the group that was stimulated 10 days after surgery. These results demonstrate that the timing of mechanical stimulation affects the physical properties of the callus and the migration of MSC to the fracture site. Published by Elsevier Inc.

Microspore culture in Corchorus olitorius: effect of growth regulators, temperature and sucrose on callus formation.

PubMed

Ali, M A; Jones, J K

2000-06-01

Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C. olitorius. The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo. Initially, 10-30% microspores divided symmetrically, but only 0.2-1% of the dividing microspores continued dividing and produced multicellular microcalli. About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA. Incubation in the dark at temperatures of 35 degrees C for 1 day and then 25 degrees C was found effective for induction of first embryonic division in Corchorus. The frequency of microspore callus formation was higher on medium containing either 3% or 5% sucrose. Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius. On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced.

Effect of vacuum-assisted closure combined with open bone grafting to promote rabbit bone graft vascularization.

PubMed

Hu, Chao; Zhang, Taogen; Ren, Bin; Deng, Zhouming; Cai, Lin; Lei, Jun; Ping, Ansong

2015-04-27

Patients with composite bone non-union and soft tissue defects are difficult to treat. Vacuum-assisted closure (VAC) combined with open bone grafting is one of the most effective treatments at present. The aim of the present study was to preliminarily investigate the effect and mechanism of VAC combined with open bone grafting to promote rabbit bone graft vascularization, and to propose a theoretical basis for clinical work. Twenty-four New Zealand white rabbits were randomly divided into an experimental and a control group. Allogeneic bones were grafted and banded with the proximal femur with a suture. The experimental group had VAC whereas the control group had normal wound closure. The bone vascularization rate was compared based on X-ray imaging, fluorescent bone labeling (labeled tetracycline hydrochloride and calcein), calcium content in the callus, and expression of fibroblast growth factor-2 (FGF-2) in bone allografts by Western blot analysis at the 4th, 8th, and 12th week after surgery. At the 4th, 8th, and 12th week after surgery, the results of the tests demonstrated that the callus was larger, contained more calcium (p<0.05), and expressed FGF-2 at higher levels (p<0.05) in the experimental group than in the control group. Fluorescent bone labeling showed the distance between the two fluorescent ribbons was significantly shorter in the control group than in the experimental group at the 8th and 12th week after surgery. VAC combined with open bone grafting promoted rabbit bone graft vascularization.

Comparative transcriptomic analysis of key genes involved in flavonoid biosynthetic pathway and identification of a flavonol synthase from Artemisia annua L.

PubMed

Liu, S; Liu, L; Tang, Y; Xiong, S; Long, J; Liu, Z; Tian, N

2017-07-01

The regulatory mechanism of flavonoids, which synergise anti-malarial and anti-cancer compounds in Artemisia annua, is still unclear. In this study, an anthocyanidin-accumulating mutant callus was induced from A. annua and comparative transcriptomic analysis of wild-type and mutant calli performed, based on the next-generation Illumina/Solexa sequencing platform and de novo assembly. A total of 82,393 unigenes were obtained and 34,764 unigenes were annotated in the public database. Among these, 87 unigenes were assigned to 14 structural genes involved in the flavonoid biosynthetic pathway and 37 unigenes were assigned to 17 structural genes related to metabolism of flavonoids. More than 30 unigenes were assigned to regulatory genes, including R2R3-MYB, bHLH and WD40, which might regulate flavonoid biosynthesis. A further 29 unigenes encoding flavonoid biosynthetic enzymes or transcription factors were up-regulated in the mutant, while 19 unigenes were down-regulated, compared with the wild type. Expression levels of nine genes involved in the flavonoid pathway were compared using semi-quantitative RT-PCR, and results were consistent with comparative transcriptomic analysis. Finally, a putative flavonol synthase gene (AaFLS1) was identified from enzyme assay in vitro and in vivo through heterogeneous expression, and confirmed comparative transcriptomic analysis of wild-type and mutant callus. The present work has provided important target genes for the regulation of flavonoid biosynthesis in A. annua. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.

Antimicrobial flavonoids from Tridax procumbens.

PubMed

Jindal, Alka; Kumar, Padma

2012-01-01

Callus culture of Tridax procumbens has been established on Murashige and Skoog's medium supplemented with NAA and BAP from nodal segments. Free and bound flavonoids were extracted from 2, 4, 6 and 8 weeks old calli by a well-established method. These free flavonoids were then screened against Staphylococcus aureus (bacteria) and Candida albicans (yeast) for their antimicrobial potential. Minimum inhibitory concentration, minimum bactericidal/fungicidal concentrations and total activity were also evaluated. Apigenin, quercetin and kaempferol were identified from free flavonoids of 4 weeks old callus (most active) through, thin layer chromatography, (TLC) preparative TLC, MP and IR spectral studies.

[Cloning,expression and functional identification of secoisolariciresinol dehydrogenase gene from Dysosma versipellis callus].

PubMed

Shen, Yun; Chen, Ri-Dao; Xie, Ke-Bo; Zou, Jian-Hua; Dai, Jun-Gui

2016-12-01

Secoisolariciresinol dehydrogenase (SDH) is a key enzyme involved in the biosynthetic pathway of podophyllotoxin.In this study, two SDH candidate genes,SO282 and SO1223, were cloned from callus of Dysosma versipellis by homology-based PCR and rapid amplification of cDNA end (RACE).The SDH candidate genes were expressed in Escherichia coli and the subsequent enzyme assay in vitro showed that recombinant SO282 had the SDH activity. These results pave the way to the follow-up investigation of the biosynthetic of podophyllotoxin. Copyright© by the Chinese Pharmaceutical Association.

Induction of secondary metabolite production by UV-C radiation in Vitis vinifera L. Öküzgözü callus cultures.

PubMed

Cetin, Emine Sema

2014-09-04

The aim of the present work was to examine the role of UV-C irradiation on the production of secondary metabolites (total phenolic, total flavanols, total flavonols, catechin, ferulic acid and trans-resveratrol in phenolic compounds and α-, β-, γ- δ-tocopherols) in callus cultures. Studies on the effects of UV-C treatment on callus culture are seldom and generally focused on UV-B. However UV-C radiation play an important role in accumule secondary metabolites. In this study, callus cultures from Öküzgözü grape cultivar were initiated from leaf petiole explants. Calli formed after 6 weeks on the medium supplemented with 0.5 mg L-1 benzylaminopurine (BA), 0.5 mg L-1 indole acetic acid (IAA) on B5 media. Callus tissues were exposed to UV-C irradiation at 10, 20 and 30 cm distances from the UV source for 5 and 10 minutes and samples were collected at hours 0, 24 and 48. The greatest total phenolic content (155.14 mg 100 g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. 24 h and 48 h incubation times, 30 cm and 5 min were the most appropriate combination of UV-C application in total flavanol content. Maximum total flavonol content (7.12 mg 100 g-1) was obtained on 0 h, 5 min and 20 cm combination. The highest (+)- catechin accumulation (8.89 mg g-1) was found in calli with 10 min UV-C application from 30 cm distance and sampled after 48 h. Ferulic acid content increased 6 fold in Öküzgözü callus cultures (31.37 μg g-1) compared to the control group. The greatest trans-resveratrol content (8.43 μg g-1) was detected in calli exposed to UV-C for 5 min from 30 cm distance and sampled after 24 h. The highest α-tocopherol concentration was found in calli exposed to UV-C for 10 min from 30 cm distance and sampled after 24 h. As a conclusion, it was showed that UV-C radiation had remarkable promoting effects on the accumulation of secondary metabolites in the calli of Öküzgözü grape cultivar.

Assessment of genetic stability and instability of tissue culture-propagated plantlets of Aloe vera L. by RAPD and ISSR markers.

PubMed

Rathore, Mangal Singh; Chikara, J; Mastan, Shaik G; Rahman, H; Anand, K G V; Shekhawat, N S

2011-11-01

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.

Zeatin and Thidiazuron Induced Embryogenic Calli From In Vitro Leaf and Stem of Jojoba (Simmondsia chinensis).

PubMed

El-Ashry, Amal Abd El-Latif; Gabr, Ahmed Mohamed Magdy; Bekheet, Shawky Abd El-Hamid

2017-01-01

Jojoba is a promising industrial plant, which recommended with pharmaceutical benefits. The present study was conducted to stimulate embryogenic calli formation from jojoba using zeatin and thidiazuron (TDZ), as well as determination of the antioxidant activity of proliferated calli. For callus induction, leaf and stem explants derived from in vitro grown shootlets, were cultured on Murashige and Skoog (MS) medium with different combinations of 0.5 mg L-1 benzyl adenine (BA) or kinetin with 2,4-Dichlorophenoxyacetic acid (2,4-D), Naphthalene acetic acid (NAA) and picloram at 0.5 or 1mg L-1. To stimulate embryogenic calli, friable callus were transferred to woody plant medium (WPM) supplemented with different concentrations of zeatin or TDZ. Antioxidant activity of different treatments was determined using hexane or petroleum ether extraction. Data was analyzed as mean±standard deviation (SD). The MS medium supplemented with 0.5 mg L-1 BA+0.5 or 1 mg L-1 picloram was the best treatment to obtain friable calli from both explants types. WPM medium supplemented with 2 mg L-1 zeatin gave the highest percentage of embryogenic calli derived from leaf explants. While the highest percentage of embryogenic calli derived from stem explants was registered using 1 or 4 mg L-1 TDZ containing medium. Embryogenic calli originated from leaves explants on 1.5 mg L-1 zeatin showed promising activity of antioxidant with hexane extraction. However, embryogenic calli originated from stem explants on 1 mg L-1 TDZ showed the highest antioxidant activity with petroleum ether extraction. TDZ has promising effect on embryogenic callus induction from stem explants. While, zeatin has promising effect on embryogenic callus induction from leaf explants.

Expression and immunogenicity of enterotoxigenic Escherichia coli heat-labile toxin B subunit in transgenic rice callus.

PubMed

Kim, Tae-Geum; Kim, Bang-Geul; Kim, Mi-Young; Choi, Jae-Kwon; Jung, Eun-Sun; Yang, Moon-Sik

2010-01-01

Enterotoxigenic Escherichia coli is one of the leading causes of diarrhea in developing countries, and the disease may be fatal in the absence of treatment. Enterotoxigenic E. coli heat-labile toxin B subunit (LTB) can be used as an adjuvant, as a carrier of fused antigens, or as an antigen itself. The synthetic LTB (sLTB) gene, optimized for plant codon usage, has been introduced into rice cells by particle bombardment-mediated transformation. The integration and expression of the sLTB gene were observed via genomic DNA PCR and western blot analysis, respectively. The binding activity of LTB protein expressed in transgenic rice callus to G(M1)-ganglioside, a receptor for biologically active LTB, was confirmed by G(M1)-ELISA. Oral inoculation of mice with lyophilized transgenic rice calli containing LTB generated significant IgG antibody titers against bacterial LTB, and the sera of immunized mice inhibited the binding of bacterial LTB to G(M1)-ganglioside. Mice orally immunized with non-transgenic rice calli failed to generate detectable anti-LTB IgG antibody titers. Mice immunized with plant-produced LTB generated higher IgG1 antibody titers than IgG2a, indicating a Th2-type immune response. Mice orally immunized with lyophilized transgenic rice calli containing LTB elicited higher fecal IgA antibody titers than mice immunized with non-transgenic rice calli. These experimental results demonstrate that LTB proteins produced in transgenic rice callus and given to mice by oral administration induce humoral and secreted antibody immune responses. We suggest that transgenic rice callus may be suitable as a plant-based edible vaccine to provide effective protection against enterotoxigenic E. coli heat-labile toxin.

Selection and characterization of glyphosate tolerance in birdsfoot trefoil (Lotus corniculatus)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boerboom, C.M.

1989-01-01

If birdsfoot trefoil (Lotus corniculatus L.) was tolerant to glyphosate (N-(phosphonomethyl)glycine), Canada thistle (Cirsium arvense (L.) Scop.) and other dicot weeds could be selectively controlled in certified seed production fields. Glyphosate tolerance in birdsfoot trefoil was identified in plants from the cultivar Leo, plants regenerated from tolerant callus, and selfed progeny of plants regenerated from callus. Plants from the three sources were evaluated in field studies for tolerance to glyphosate at rates up to 1.6 kg ae/ha. Plants of Leo selected for tolerance exhibited a twofold range in the rate required to reduce shoot weight 50% (I{sub 50}s from 0.6more » to 1.2 kg/ha glyphosate). Plants regenerated from tolerant callus had tolerance up to 66% greater than plants regenerated from unselected callus. Transgressive segregation for glyphosate tolerance was observed in the selfed progeny of two regenerated plants that both had I{sub 50}s of 0.7 kg/ha glyphosate. The selfed progeny ranged from highly tolerant (I{sub 50} of 1.5 kg/ha) to susceptible (I{sub 50} of 0.5 kg/ha). Spray retention, {sup 14}C-glyphosate absorption and translocation did not account for the differential tolerance of nine plants that were evaluated from the three sources. The specific activity of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase ranged from 1.3 to 3.5 nmol/min{sm bullet}mg among the nine plants and was positively correlated with glyphosate tolerance. Leo birdsfoot trefoil was found to have significant variation in glyphosate tolerance which made it possible to initiate a recurrent selection program to select for glyphosate tolerance in birdsfoot trefoil. Two cycles of selection for glyphosate tolerance were practiced in three birdsfoot trefoil populations, Leo, Norcen, and MU-81.« less

Effective salt criteria in callus-cultured tomato genotypes.

PubMed

Dogan, Mahmut; Tipirdamaz, Rukiye; Demir, Yavuz

2010-01-01

Na+, Cl-, K+, Ca2+, and proline contents, the rate of lipid peroxidation level in terms of malondialdehyde (MDA) and chlorophyll content, and the changes in the activity of antioxidant enzymes, such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), ascorbate peroxidase (APX: EC 1.11.1.11), and glutathione reductase (GR: EC 1.6.4.2), in tissues of five tomato cultivars in salt tolerance were investigated in a callus culture. The selection of effective parameters used in these tomato genotypes and to find out the use of in vitro tests in place of in vivo salt tolerance tests were investigated. As a material, five different tomato genotypes during a 10-day time period were used, and 150 mM NaCl was applied at callus plant tissue. The exposure to NaCl induced a significant increase in MDA content in both salt-resistant and salt-sensitive cultivars. But the MDA content was higher in salt-sensitive cultivars. The chlorophyll content was more decreased in salt-sensitive than in salt-resistant ones. The proline amount was more increased in salt-sensitive than in salt-resistant ones. It has been reported that salt-tolerant plants, besides being able to regulate the ion and water movements, also exhibit a strong antioxidative enzyme system for effective removal of ROS. The degree of damage depends on the balance between the formation of ROS and its removal by the antioxidative scavenging system that protects against them. Exclusion or inclusion of Na+, Cl-, K+, and Ca2+, antioxidant enzymes and MDA concentration play a key protective role against stress, and this feature at the callus plant tissue used as an identifier for tolerance to salt proved to be an effective criterion.

Some ethylene biosynthesis and AP2/ERF genes reveal a specific pattern of expression during somatic embryogenesis in Hevea brasiliensis

PubMed Central

2012-01-01

Background Ethylene production and signalling play an important role in somatic embryogenesis, especially for species that are recalcitrant in in vitro culture. The AP2/ERF superfamily has been identified and classified in Hevea brasiliensis. This superfamily includes the ERFs involved in response to ethylene. The relative transcript abundance of ethylene biosynthesis genes and of AP2/ERF genes was analysed during somatic embryogenesis for callus lines with different regeneration potential, in order to identify genes regulated during that process. Results The analysis of relative transcript abundance was carried out by real-time RT-PCR for 142 genes. The transcripts of ERFs from group I, VII and VIII were abundant at all stages of the somatic embryogenesis process. Forty genetic expression markers for callus regeneration capacity were identified. Fourteen markers were found for proliferating calli and 35 markers for calli at the end of the embryogenesis induction phase. Sixteen markers discriminated between normal and abnormal embryos and, lastly, there were 36 markers of conversion into plantlets. A phylogenetic analysis comparing the sequences of the AP2 domains of Hevea and Arabidopsis genes enabled us to predict the function of 13 expression marker genes. Conclusions This first characterization of the AP2/ERF superfamily in Hevea revealed dramatic regulation of the expression of AP2/ERF genes during the somatic embryogenesis process. The gene expression markers of proliferating callus capacity to regenerate plants by somatic embryogenesis should make it possible to predict callus lines suitable to be used for multiplication. Further functional characterization of these markers opens up prospects for discovering specific AP2/ERF functions in the Hevea species for which somatic embryogenesis is difficult. PMID:23268714

Correlation of different spectral lights with biomass accumulation and production of antioxidant secondary metabolites in callus cultures of medicinally important Prunella vulgaris L.

PubMed

Fazal, Hina; Abbasi, Bilal Haider; Ahmad, Nisar; Ali, Syed Shujait; Akbar, Fazal; Kanwal, Farina

2016-06-01

Light is one of the key elicitors that directly fluctuates plant developmental processes and biosynthesis of secondary metabolites. In this study, the effects of various spectral lights on biomass accumulation and production of antioxidant secondary metabolites in callus cultures of Prunella vulgaris were investigated. Among different spectral lights, green light induced the maximum callogenic response (95%). Enhanced fresh biomass accumulation was observed in log phases on day-35, when callus cultures were exposed to yellow and violet lights. Yellow light induced maximum biomass accumulation (3.67g/100ml) from leaf explants as compared to control (1.27g/100ml). In contrast, violet lights enhanced biomass accumulation (3.49g/100ml) from petiole explant. Maximum total phenolics content (TPC; 23.9mg/g-DW) and total flavonoids content (TFC; 1.65mg/g-DW) were observed when cultures were grown under blue lights. In contrast, green and yellow lights enhanced total phenolics production (TPP; 112.52g/100ml) and total flavonoids production (TFP; 9.64g/100ml) as compared to control. The calli grown under green, red and blue lights enhanced DPPH-free radical scavenging activity (DFRSA; 91.3%, 93.1% and 93%) than control (56.44%) respectively. The DFRSA was correlated either with TPC and TFC or TPP and TFP. Furthermore, yellow lights enhanced superoxide dismutase (SOD), peroxidase (POD) and protease activities, however, the content of total protein (CTP) was higher in control cultures (186μg BSAE/mg FW) as compared to spectral lights. These results suggest that the exposure of callus cultures to various spectral lights have shown a key role in biomass accumulation and production of antioxidant secondary metabolites. Copyright © 2016 Elsevier B.V. All rights reserved.

FLUOXETINE INHIBITS OSTEOBLAST DIFFERENTIATION & MINERALIZATION IN FRACTURE HEALING

PubMed Central

Bradaschia-Correa, Vivian; Josephson, Anne M; Mehta, Devan; Mizrahi, Matthew; Neibart, Shane S; Liu, Chao; Kennedy, Oran; Castillo, Alesha B; Egol, Kenneth A; Leucht, Philipp

2016-01-01

Chronic use of selective serotonin reuptake inhibitors (SSRIs) for the treatment of depression has been linked to osteoporosis. In this study, we investigated the effect of chronic SSRI use on fracture healing in two murine models of bone regeneration. First, we performed a comprehensive analysis of endochondral bone healing in a femur fracture model. C57/BL6 mice treated with fluoxetine, the most commonly prescribed SSRI, developed a normal cartilaginous soft-callus at 14 days after fracture and demonstrated a significantly smaller and biomechanically weaker bony hard-callus at 28 days. In order to further dissect the mechanism that resulted in a smaller bony regenerate, we used an intramembranous model of bone healing and revealed that fluoxetine treatment resulted in a significantly smaller bony callus at 7 and 14 days postinjury. In order to test whether the smaller bony regenerate following fluoxetine treatment was caused by an inhibition of osteogenic differentiation and/or mineralization, we employed in vitro experiments, which established that fluoxetine treatment decreases osteogenic differentiation and mineralization and that this effect is serotonin-independent. Finally, in a translational approach, we tested whether cessation of the medication would result in restoration of the regenerative potential. However, histologic and µCT analysis revealed non-union formation in these animals with fibrous tissue interposition within the callus. In conclusion, fluoxetine exerts a direct, inhibitory effect on osteoblast differentiation and mineralization, shown in two disparate murine models of bone repair. Discontinuation of the drug did not result in restoration of the healing potential, but rather led to complete arrest of the repair process. Besides the well-established effect of SSRIs on bone homeostasis, our study provides strong evidence that fluoxetine use negatively impacts fracture healing. PMID:27869327

[18S-25S rDNA variation in tissue culture of some Gentiana L. species].

PubMed

Mel'nyk, V M; Andrieiev, I O; Spiridonova, K V; Strashniuk, N M; Kunakh, V A

2007-01-01

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation.

PubMed

García-Rodríguez, J; Martínez-Reina, J

2017-02-01

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions' orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young's modulus [Formula: see text] GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young's modulus in the preferential direction of 9-16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.

Microprojectile Bombardment Transformation of Date Palm Using the Insecticidal Cholesterol Oxidase (ChoA) Gene.

PubMed

Allam, Mai A; Saker, Mahmoud M

2017-01-01

The overall objective of this work is to optimize the transformation system for date palm as a first step toward production of date palm clones resistant to noxious pests. A construct harboring the cholesterol oxidase (ChoA) gene, which renders plant resistance against insect attack, is introduced into embryogenic date palm callus using the PDS-1000/He particle bombardment system. The process involves the establishment of embryogenic callus cultures as well as immature embryo-derived microcalli that are used as target tissues for shooting and optimization of transformation conditions. This chapter in addition explains molecular and histochemical assays conducted to confirm gene integration and expression.

Biotechnology and genetic optimization of fast-growing hardwoods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garton, S.; Syrkin-Wurtele, E.; Griffiths, H.

1991-02-01

A biotechnology research program was initiated to develop new clones of fast-growing Populus clones resistant to the herbicide glyphosate and resistant to the leaf-spot and canker disease caused by the fungus Septoria musiva. Glyphosate-resistant callus was selected from stem segments cultured in vitro on media supplemented with the herbicide. Plants were regenerated from the glyphosate-resistant callus tissue. A portion of plants reverted to a glyphosate susceptible phenotype during organogenesis. A biologically active filtrate was prepared from S. musiva and influenced fresh weight of Populus callus tissue. Disease-resistant plants were produced through somaclonal variation when shoots developed on stem internodes culturedmore » in vitro. Plantlets were screened for disease symptoms after spraying with a suspension of fungal spores. A frequency of 0.83 percent variant production was observed. Genetically engineered plants were produced after treatment of plant tissue with Agrobacterium tumefasciens strains carrying plasmid genes for antibiotic resistance. Transformers were selected on media enriched with the antibiotic, kanamycin. Presence of foreign DNA was confirmed by Southern blot analysis. Protoplasts of popular were produced but did not regenerate into plant organs. 145 refs., 12 figs., 36 tabs.« less

ED-XRF spectrometry-based comparative inorganic profile of leaf-derived in vitro calli and in vivo leaf samples of Phyllanthus amarus Schum. & Thonn.--a hepatoprotective herb.

PubMed

Nayak, P; Behera, P R; Thirunavoukkarasu, M; Chand, P K

2011-03-01

The Energy Dispersive X-ray Fluorescence (ED-XRF) set-up incorporating a molybdenum secondary exciter was used for quantitative determination of major and minor elements in leaves of in vivo grown medicinal herb Phyllanthus amarus vis-á-vis its leaf-derived in vitro callus culture. The elements such as K, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr and Pb were identified, quantified and compared between both the sources. Experimental results revealed that, compared to the naturally grown herb, in vitro leaf-derived callus cultures were more efficient in accumulating inorganic elements, especially trace elements, which are essential for growth and development and more importantly for prevention and cure of diseases. This investigation on a medicinal plant species is the first of its kind to have used the ED-XRF technique to demonstrate a comparative account of the elemental profile of in vitro callus cultures with their in vivo donor in order to explore the possibility of exploiting the former as a viable alternative and a renewable source of phytochemicals. Copyright © 2010 Elsevier Ltd. All rights reserved.

Characterising the biophysical properties of normal and hyperkeratotic foot skin.

PubMed

Hashmi, Farina; Nester, Christopher; Wright, Ciaran; Newton, Veronica; Lam, Sharon

2015-01-01

Plantar foot skin exhibits unique biophysical properties that are distinct from skin on other areas of the body. This paper characterises, using non-invasive methods, the biophysical properties of foot skin in healthy and pathological states including xerosis, heel fissures, calluses and corns. Ninety three people participated. Skin hydration, elasticity, collagen and elastin fibre organisation and surface texture was measured from plantar calluses, corns, fissured heel skin and xerotic heel skin. Previously published criteria were applied to classify the severity of each skin lesion and differences in the biophysical properties compared between each classification. Calluses, corns, xerotic heel skin and heel fissures had significantly lower levels of hydration; less elasticity and greater surface texture than unaffected skin sites (p < 0.01). Some evidence was found for a positive correlation between hydration and elasticity data (r ≤ 0.65) at hyperkeratotic sites. Significant differences in skin properties (with the exception of texture) were noted between different classifications of skin lesion. This study provides benchmark data for healthy and different severities of pathological foot skin. These data have applications ranging from monitoring the quality of foot skin, to measuring the efficacy of therapeutic interventions.

A controlled trial of glutamine effects on bone healing.

PubMed

Polat, Onur; Kilicoglu, Sibel Serin; Erdemli, Esra

2007-01-01

Glutamine is considered a nonessential amino acid, but it may be conditionally essential in patients with catabolic conditions. For centuries, researchers have looked for ways to promote and accelerate fracture healing. This controlled animal study examines the effects of glutamine on fracture healing. The left tibias of 10 standardized albino rats were broken at the distal third to produce a closed fracture. L-glutamine/L-alanyl solution (2.0 mL/kg) was administered through the tail veins of half the rats for the first 7 d, and physiologic serum alone was given to the control group. On the 21st day, all rats were euthanized and their left legs removed; after histologic observation, the tibias were examined under light microscopy. In the glutamine-injected group, development of primary callus was quicker and more regular than in the control group. The control group produced insufficient fibrous callus, and the glutamine group attained formed cartilaginous callus. Glutamine was noted to have positive effects on healing of traumatically fractured bone through attainment of positive nitrogen balance. This effect was minimal in enhancing the quality of fracture healing under conditions of stress, but some effect was noted on the speed of healing. Further research is needed in this area.

Influence of functionally graded pores on bone ingrowth in cementless hip prosthesis: a finite element study using mechano-regulatory algorithm.

PubMed

Tarlochan, Faris; Mehboob, Hassan; Mehboob, Ali; Chang, Seung-Hwan

2018-06-01

Cementless hip prostheses with porous outer coating are commonly used to repair the proximally damaged femurs. It has been demonstrated that stability of prosthesis is also highly dependent on the bone ingrowth into the porous texture. Bone ingrowth is influenced by the mechanical environment produced in the callus. In this study, bone ingrowth into the porous structure was predicted by using a mechano-regulatory model. Homogenously distributed pores (200 and 800 [Formula: see text]m in diameter) and functionally graded pores along the length of the prosthesis were introduced as a porous coating. Bone ingrowth was simulated using 25 and 12 [Formula: see text]m micromovements. Load control simulations were carried out instead of traditionally used displacement control. Spatial and temporal distributions of tissues were predicted in all cases. Functionally graded pore decreasing models gave the most homogenous bone distribution, the highest bone ingrowth (98%) with highest average Young's modulus of all tissue phenotypes approximately 4.1 GPa. Besides this, the volume of the initial callus increased to 8.33% in functionally graded pores as compared to the 200 [Formula: see text]m pore size models which increased the bone volume. These findings indicate that functionally graded porous surface promote bone ingrowth efficiently which can be considered to design of surface texture of hip prosthesis.

Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cahoon, E.B.; Shanklin, J.; Ohlrogge, J.B.

1992-12-01

Little is known about the metabolic origin of petroselinic acid (18:1[Delta][sup 6cis]), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species. To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the [Delta][sup 9]-stearoyl-ACP desaturase of avocado. In these extracts, proteins of 39 and 36 kDa were detected. Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid. A cDNA encoding the 36-kDa peptide was isolated from a coriandermore » endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and [Delta][sup 4]-hexadecenoic acid, both of which were absent from control callus. These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase. 27 refs., 5 figs.« less

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant.

PubMed

Syamala, D; Devi, Prathibha

2003-12-01

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.

Bermudagrass (Cynodon spp.).

PubMed

Ge, Yaxin; Wang, Zeng-Yu

2006-01-01

Bermudagrass is an important warm-season forage and turf species widely grown in the southern United States. This chapter describes a rapid and efficient protocol that allows for the generation of a large number of transgenic bermudagrass plants, bypassing the callus formation phase. Stolon nodes are infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA binary vectors. Hygromycin phosphotransferase gene (hph) is used as the selectable marker and hygromycin is used as the selection agent. Green shoots are directly produced from infected stolon nodes 4 to 5 wk after hygromycin selection. Without callus formation and with minimum tissue culture, this procedure allowed us to obtain well-rooted transgenic plantlets in only 7 wk and greenhouse-grown plants in only 9 wk.

Bioreactor Steroid Production and Analysis of Date Palm Embryogenic Callus.

PubMed

El-Sharabasy, Sherif; El-Dawayati, Maiada

2017-01-01

Several compounds and families of compounds of date palm secondary metabolites have been investigated. The analysis of date palm tissue has shown the abundance of secondary metabolites including phytosterols, e.g., steroids, an important group of pharmaceutical compounds. Biotechnology offers the opportunity to utilize cells, tissues, and organs grown in vitro and manipulated to obtain desired compounds. This chapter presents a protocol for the production, determination, and identification of steroids in date palm callus tissue. The addition of 0.01 mg/L pyruvic acid as a precursor to MS liquid culture medium enhances steroid production. In addition, the chapter describes the sterol analytical techniques based on gas-liquid chromatography and gas chromatography-mass spectrometry.

Preliminary molecular detection of the somatic embryogenesis receptor-like kinase (VpSERK) and knotted-like homeobox (VpKNOX1) genes during in vitro morphogenesis of Vanilla planifolia Jacks.

PubMed

Ramírez-Mosqueda, Marco A; Iglesias-Andreu, Lourdes G; Sáenz, Luis; Córdova, Iván

2018-02-01

This work aimed to evaluate the embryogenic competence of different tissues from different stages (friable callus, bud-regenerating callus, and whole buds) of Vanilla planifolia , through the molecular detection of the somatic embryogenesis receptor-like kinase ( VpSERK ) and knotted-like homeobox ( VpKNOX1 ) genes. RNA was extracted with Trizol ® , cDNA was obtained, and the studied transcripts were amplified. Using non-specific primers, VpSERK and VpSTM gene expression was detected in the three stages evaluated. This study might contribute to providing an explanation for the recalcitrance of this Vanilla species to somatic embryogenesis.

Cutin plays a role in differentiation of endosperm-derived callus of kiwifruit.

PubMed

Popielarska-Konieczna, Marzena; Kozieradzka-Kiszkurno, Małgorzata; Bohdanowicz, Jerzy

2011-11-01

Cutin fluorescence, after auramine O treatment, was detected on the surface of organogenic areas (protuberances) of endosperm derived callus induced on Murashige and Skoog medium with thidiazuron (0.5 mg l(-1)) in darkness. Electron micrographs of the protuberances revealed cuticle, visible as a dark-staining layer, and amorphous waxes on the cell wall. In some cases the cells of the epidermis-like layer and shoot buds at early stages of development showed thick and characteristically wavy cutin. This waviness corresponds with the wrinkled appearance of the cell wall as observed by scanning electron microscopy. The role of multivesicular bodies in cutin production and transfer to the plasma membrane is discussed.

Determination of Suitable Microspore Stage and Callus Induction from Anthers of Kenaf (Hibiscus cannabinus L.)

PubMed Central

Binti Kayat, Fatimah; Ermiena Surya Mat Hussin, Zeti; Susanto, Dwi; Ariffulah, Mohammed

2014-01-01

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6–8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA. PMID:24757416

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

PubMed

Raji, Jennifer A; Frame, Bronwyn; Little, Daniel; Santoso, Tri Joko; Wang, Kan

2018-01-01

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

An Efficient Plant Regeneration and Transformation System of Ma Bamboo (Dendrocalamus latiflorus Munro) Started from Young Shoot as Explant

PubMed Central

Ye, Shanwen; Cai, Changyang; Ren, Huibo; Wang, Wenjia; Xiang, Mengqi; Tang, Xiaoshan; Zhu, Caiping; Yin, Tengfei; Zhang, Li; Zhu, Qiang

2017-01-01

Genetic engineering technology has been successfully used in many plant species, but is limited in woody plants, especially in bamboos. Ma bamboo (Dendrocalamus latiflorus Munro) is one of the most important bamboo species in Asia, and its genetic improvement was largely restricted by the lack of an efficient regeneration and transformation method. Here we reported a plantlet regeneration and Agrobacterium-mediated transformation protocol by using Ma bamboo young shoots as explants. Under our optimized conditions, embryogenic calluses were successfully induced from the excised young shoots on callus induction medium and rapidly grew on callus multiplication medium. Shoots and roots were regenerated on shoot induction medium and root induction medium, respectively, with high efficiency. An Agrobacterium-mediated genetic transformation protocol of Ma bamboo was established, verified by PCR and GUS staining. Furthermore, the maize Lc gene under the control of the ubiquitin promoter was successfully introduced into Ma bamboo genome and generated an anthocyanin over-accumulation phenotype. Our methods established here will facilitate the basic research as well as genetic breeding of this important bamboo species. Key achievements: A stable and high efficiency regeneration and Agrobacterium-mediated transformation protocol for Ma bamboo from vegetative organ is established. PMID:28798758

Influence of auxins combinations on accumulation of reserpine in the callus of Rauvolfia tetraphylla L.

PubMed

Anitha, S; Kumari, B D Ranjitha

2007-11-01

Reserpine is a monoterpene indole alkaloid used to treat hypertension because of its hypotensive property and psychiatric disorders because of its tranquilizing effect. Protocol has been standardized to enhance the synthesis of reserpine in leaf derived calli of Rauvolfia tetraphylla L. by adjusting the auxins combinations in the medium consisting of MS nutrient salts and B5 vitamins. Auxins such as naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were used in 1-5 microM concentration along with 9 microM concentration of 2,4 dichlorophenoxy acetic acid (2,4-D), which was found suitable for callus induction. The combination of (2,4-D) with NAA had been proved to accumulate maximum amount of reserpine followed by 2,4-D with IBA. The IAA with 2,4-D combination yielded very less amount of reserpine than the other combinations and 9 microM 2,4-D alone. The results suggest that there may be synergetic effect of NAA with 2,4-D and IBA with 2,4-D for increase in the biomass and reserpine accumulation and antagonistic effect of IAA with 2,4-D for the above said factors in the callus.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants.

PubMed

Rodríguez López, Carlos M; Wetten, Andrew C; Wilkinson, Michael J

2010-06-01

*Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. *For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. *Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. *Statistical analysis confirmed this rather counterintuitive finding that leaves of 'late regenerants' exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

Mutation of a Rice Gene Encoding a Phenylalanine Biosynthetic Enzyme Results in Accumulation of Phenylalanine and Tryptophan[W

PubMed Central

Yamada, Tetsuya; Matsuda, Fumio; Kasai, Koji; Fukuoka, Shuichi; Kitamura, Keisuke; Tozawa, Yuzuru; Miyagawa, Hisashi; Wakasa, Kyo

2008-01-01

Two distinct biosynthetic pathways for Phe in plants have been proposed: conversion of prephenate to Phe via phenylpyruvate or arogenate. The reactions catalyzed by prephenate dehydratase (PDT) and arogenate dehydratase (ADT) contribute to these respective pathways. The Mtr1 mutant of rice (Oryza sativa) manifests accumulation of Phe, Trp, and several phenylpropanoids, suggesting a link between the synthesis of Phe and Trp. Here, we show that the Mtr1 mutant gene (mtr1-D) encodes a form of rice PDT with a point mutation in the putative allosteric regulatory region of the protein. Transformed callus lines expressing mtr1-D exhibited all the characteristics of Mtr1 callus tissue. Biochemical analysis revealed that rice PDT possesses both PDT and ADT activities, with a preference for arogenate as substrate, suggesting that it functions primarily as an ADT. The wild-type enzyme is feedback regulated by Phe, whereas the mutant enzyme showed a reduced feedback sensitivity, resulting in Phe accumulation. In addition, these observations indicate that rice PDT is critical for regulating the size of the Phe pool in plant cells. Feeding external Phe to wild-type callus tissue and seedlings resulted in Trp accumulation, demonstrating a connection between Phe accumulation and Trp pool size. PMID:18487352

A microdroplet cell culture based high frequency somatic embryogenesis system for pigeonpea, Cajanus cajan (L.) Millsp.

PubMed

Kumar, Nagan Udhaya; Gnanaraj, Muniraj; Sindhujaa, Vajravel; Viji, Maluventhen; Manoharan, Kumariah

2015-09-01

A protocol for high frequency production of somatic embryos was worked out in pigeonpea, Cajanus cajan (L.) Millsp. The protocol involved sequential employment of embryogenic callus cultures, low density cell suspension cultures and a novel microdroplet cell culture system. The microdroplet cell cultures involved culture of a single cell in 10 μI of Murashige and Skoog's medium supplemented with phytohormones, growth factors and phospholipid precursors. By employing the microdroplet cell cultures, single cells in isolation were grown into cell clones which developed somatic embryos. Further, 2,4-dichlorophenoxyacetic acid, kinetin, polyethylene glycol, putrescine, spermine, spermidine, choline chloride, ethanolamine and LiCl were supplemented to the low density cell suspension cultures and microdroplet cell cultures to screen for their cell division and somatic embryogenesis activity. Incubation of callus or the inoculum employed for low density cell suspension cultures and microdroplet cell cultures with polyethylene glycol was found critical for induction of somatic embryogenesis. Somatic embryogenesis at a frequency of 1.19, 3.16 and 6.51 per 10(6) cells was achieved in the callus, low density cell suspension cultures and microdroplet cell cultures, respectively. Advantages of employing microdroplet cell cultures for high frequency production of somatic embryos and its application in genetic transformation protocols are discussed.

Relationship between endogenous hormonal content and somatic organogenesis in callus of peach (Prunus persica L. Batsch) cultivars and Prunus persica×Prunus dulcis rootstocks.

PubMed

Pérez-Jiménez, Margarita; Cantero-Navarro, Elena; Pérez-Alfocea, Francisco; Le-Disquet, Isabel; Guivarc'h, Anne; Cos-Terrer, José

2014-05-01

The relationship between endogenous hormones content and the induction of somatic peach plant was studied. To induce multiple shoots from callus derived from the base of stem explants of the scion cultivars 'UFO-3', 'Flariba' and 'Alice Bigi', and the peach×almond rootstocks 'Garnem' and 'GF677', propagated plants were cultured on Murashige and Skoog salts augmented with 0.1mgL(-1) of indolebutyric acid, 1mgL(-1) of 6-benzylaminopurine and 3% sucrose. The highest regeneration rate was obtained with the peach×almond rootstocks. Endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), zeatin (Z), zeatin riboside (ZR), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA) were analyzed in the organogenic callus. Lower levels of several hormones, namely Z, ZR, ABA, and ACC were found in the peach×almond rootstock compared to peach cultivars, while IAA and SA presented inconclusive returns. These results suggest that the difference in somatic organogenesis capacity observed in peach and peach×almond hybrids is markedly affected by the endogenous hormonal content of the studied genotypes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects.

PubMed

Cipriano, Jamile L D; Cruz, Ana Cláudia F; Mancini, Karina C; Schmildt, Edilson R; Lopes, José Carlos; Otoni, Wagner C; Alexandre, Rodrigo S

2018-01-01

The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

Genetic Transformation of Metroxylon sagu (Rottb.) Cultures via Agrobacterium-Mediated and Particle Bombardment

PubMed Central

Ibrahim, Evra Raunie

2014-01-01

Sago palm (Metroxylon sagu) is a perennial plant native to Southeast Asia and exploited mainly for the starch content in its trunk. Genetic improvement of sago palm is extremely slow when compared to other annual starch crops. Urgent attention is needed to improve the sago palm planting material and can be achieved through nonconventional methods. We have previously developed a tissue culture method for sago palm, which is used to provide the planting materials and to develop a genetic transformation procedure. Here, we report the genetic transformation of sago embryonic callus derived from suspension culture using Agrobacterium tumefaciens and gene gun systems. The transformed embryoids cells were selected against Basta (concentration 10 to 30 mg/L). Evidence of foreign genes integration and function of the bar and gus genes were verified via gene specific PCR amplification, gus staining, and dot blot analysis. This study showed that the embryogenic callus was the most suitable material for transformation as compared to the fine callus, embryoid stage, and initiated shoots. The gene gun transformation showed higher transformation efficiency than the ones transformed using Agrobacterium when targets were bombarded once or twice using 280 psi of helium pressure at 6 to 8 cm distance. PMID:25295258

Fracture healing in mice under controlled rigid and flexible conditions using an adjustable external fixator.

PubMed

Röntgen, Viktoria; Blakytny, Robert; Matthys, Romano; Landauer, Mario; Wehner, Tim; Göckelmann, Melanie; Jermendy, Philipp; Amling, Michael; Schinke, Thorsten; Claes, Lutz; Ignatius, Anita

2010-11-01

Mice are increasingly used to investigate mechanobiology in fracture healing. The need exists for standardized models allowing for adjustment of the mechanical conditions in the fracture gap. We introduced such a model using rigid and flexible external fixators with considerably different stiffness (axial stiffnesses of 18.1 and 0.82 N/mm, respectively). Both fixators were used to stabilize a 0.5 mm osteotomy gap in the femur of C57BL/6 mice (each n = 8). Three-point bending tests, µCT, and histomorphometry demonstrated a different healing pattern after 21 days. Both fixations induced callus formation with a mixture of intramembranous and enchondral ossification. Under flexible conditions, the bending stiffness of the callus was significantly reduced, and a larger but qualitatively inferior callus with a significantly lower fraction of bone but a higher fraction of cartilage and soft tissue was formed. Monitoring of the animal movement and the ground reaction forces demonstrated physiological loading with no significant differences between the groups, suggesting that the differences in healing were not based on a different loading behavior. In summary, flexible external fracture fixation of the mouse femur led to delayed fracture healing in comparison to a more rigid situation. © 2010 Orthopaedic Research Society.

Transfer of the second to the first metatarsal ray in a case of lawn mower injury: a case report.

PubMed

Sassu, Paolo; Tsai, Tsu Min

2009-01-01

The medial longitudinal arch of the foot plays a major role for a physiologic transfer of the load from the heel to the forefoot during walking and running. Traumatic amputation that involve either the great toe or the whole first metatarsal bone can lead to collapse of the medial longitudinal arch, overload of the metatarsal heads, and painful callus formation. If replant of the amputated part is not possible or has failed, it is advisable to reconstruct the medial longitudinal arch in order to re-establish a functional transfer of the load in the foot. We present a case of a young lady who suffered from traumatic amputation at the distal third of the first metatarsal. Replantation failed due to the severity of the initial injury. Despite a good coverage of the defect with a lateral arm flap, the patient developed a painful plantar callus underneath the amputated stump. The adjacent second metatarsal ray was then raised as a pedicled flap including bone and soft tissues and transferred to the first ray in order to reconstruct a physiologic medial longitudinal arch. The patient had excellent functional results with no recurrence of the callus. (c) 2008 Wiley-Liss, Inc.

Determination of suitable microspore stage and callus induction from anthers of kenaf (Hibiscus cannabinus L.).

PubMed

Ibrahim, Ahmed Mahmood; Kayat, Fatimah Binti; Hussin, Zeti Ermiena Surya Mat; Susanto, Dwi; Ariffulah, Mohammed

2014-01-01

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.

Cell wall composition throughout development for the model grass Brachypodium distachyon

PubMed Central

Rancour, David M.; Marita, Jane M.; Hatfield, Ronald D.

2012-01-01

Temperate perennial grasses are important worldwide as a livestock nutritive energy source and a potential feedstock for lignocellulosic biofuel production. The annual temperate grass Brachypodium distachyon has been championed as a useful model system to facilitate biological research in agriculturally important temperate forage grasses based on phylogenetic relationships. To physically corroborate genetic predictions, we determined the chemical composition profiles of organ-specific cell walls throughout the development of two common diploid accessions of Brachypodium distachyon, Bd21-3 and Bd21. Chemical analysis was performed on cell walls isolated from distinct organs (i.e., leaves, sheaths, stems, and roots) at three developmental stages of (1) 12-day seedling, (2) vegetative-to-reproductive transition, and (3) mature seed fill. In addition, we have included cell wall analysis of embryonic callus used for genetic transformations. Composition of cell walls based on components lignin, hydroxycinnamates, uronosyls, neutral sugars, and protein suggests that Brachypodium distachyon is similar chemically to agriculturally important forage grasses. There were modest compositional differences in hydroxycinnamate profiles between accessions Bd21-3 and Bd21. In addition, when compared to agronomical important C3 grasses, more mature Brachypodium stem cell walls have a relative increase in glucose of 48% and a decrease in lignin of 36%. Though differences exist between Brachypodium and agronomical important C3 grasses, Brachypodium distachyon should be still a useful model system for genetic manipulation of cell wall composition to determine the impact upon functional characteristics such as rumen digestibility or energy conversion efficiency for bioenergy production. PMID:23227028

Corns and Calluses

MedlinePlus

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Determination of abscisic acid and its glucosyl ester in embryogenic callus cultures of Vitis vinifera in relation to the maturation of somatic embryos using a new liquid chromatography-ELISA analysis method.

PubMed

Prado, María Jesús; Largo, Asier; Domínguez, Cristina; González, María Victoria; Rey, Manuel; Centeno, María Luz

2014-06-15

The levels of abscisic acid (ABA), its conjugate ABA-GE, and IAA were determined in embryogenic calli of Vitis vinifera L. (cv. Mencía) cultured in DM1 differentiation medium, to relate them to the maturation process of somatic embryos. To achieve this goal, we developed an analytical method that included two steps of solid-phase extraction, chromatographic separation by HPLC, ABA-GE hydrolysis, and sensitive ELISA quantification. Because the ABA immunoassay was based on new polyclonal antibodies raised against a C4'-ABA conjugate, the assay was characterized (detection limit, midrange, measure range, and cross-reaction) and validated by a comparison of the ABA data obtained with this ELISA procedure and with a physicochemical method (LC-ESI-MS/MS). Radioactive-labeled internal standards were initially added to callus extracts to correct the losses of plant hormones, and thus assure the accuracy of the measurements. The endogenous concentration of ABA in the embryogenic callus cultured in DM1 medium was doubled at the fifth week of culture, concurring with the maturation process of somatic embryos, as indicated by the accumulation of carbohydrates observed through histological analysis. The ABA-GE content was higher than ABA, decreasing at 21 days of culture in DM1 medium but increasing thereafter. The data suggest the involvement of the synthesis and conjugation of ABA in the final stages of development in grapevine somatic embryos from embryogenic callus. IAA levels were low, suggesting that auxin plays no significant role during the maturation of somatic embryos. In addition, the lower ABA levels in calli cultured in DM differentiation medium with PGRs, a medium presenting high precocious germination and deficiencies in somatic embryo development indicate that an increase in ABA content during the development of somatic embryos in grapevine is necessary for their correct maturation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures

PubMed Central

2011-01-01

Background Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica. Results We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization. Conclusion Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes. PMID:21595964

Comparison between Different Methods for Biomechanical Assessment of Ex Vivo Fracture Callus Stiffness in Small Animal Bone Healing Studies

PubMed Central

Steiner, Malte; Volkheimer, David; Meyers, Nicholaus; Wehner, Tim; Wilke, Hans-Joachim; Claes, Lutz; Ignatius, Anita

2015-01-01

For ex vivo measurements of fracture callus stiffness in small animals, different test methods, such as torsion or bending tests, are established. Each method provides advantages and disadvantages, and it is still debated which of those is most sensitive to experimental conditions (i.e. specimen alignment, directional dependency, asymmetric behavior). The aim of this study was to experimentally compare six different testing methods regarding their robustness against experimental errors. Therefore, standardized specimens were created by selective laser sintering (SLS), mimicking size, directional behavior, and embedding variations of respective rat long bone specimens. For the latter, five different geometries were created which show shifted or tilted specimen alignments. The mechanical tests included three-point bending, four-point bending, cantilever bending, axial compression, constrained torsion, and unconstrained torsion. All three different bending tests showed the same principal behavior. They were highly dependent on the rotational direction of the maximum fracture callus expansion relative to the loading direction (creating experimental errors of more than 60%), however small angular deviations (<15°) were negligible. Differences in the experimental results between the bending tests originate in their respective location of maximal bending moment induction. Compared to four-point bending, three-point bending is easier to apply on small rat and mouse bones under realistic testing conditions and yields robust measurements, provided low variation of the callus shape among the tested specimens. Axial compressive testing was highly sensitive to embedding variations, and therefore cannot be recommended. Although it is experimentally difficult to realize, unconstrained torsion testing was found to be the most robust method, since it was independent of both rotational alignment and embedding uncertainties. Constrained torsional testing showed small errors (up to 16.8%, compared to corresponding alignment under unconstrained torsion) due to a parallel offset between the specimens’ axis of gravity and the torsional axis of rotation. PMID:25781027

Evaluation of the Effects of Photobiomodulation on Partial Osteotomy in Streptozotocin-Induced Diabetes in Rats.

PubMed

Mostafavinia, Ataroalsadat; Masteri Farahani, Reza; Abdollahifar, Mohammad-Amin; Ghatrehsamani, Mahdi; Ghoreishi, Seyed Kamran; Hajihossainlou, Behnam; Chien, Sufan; Dadras, Sara; Rezaei, Fatemehalsadat; Bayat, Mohammad

2018-05-31

We examined the effects of photobiomodulation (PBM) on stereological parameters, and gene expression of Runt-related transcription factor 2 (RUNX2), osteocalcin, and receptor activator of nuclear factor kappa-B ligand (RANKL) in repairing tissue of tibial bone defect in streptozotocin (STZ)-induced type 1 diabetes mellitus (TIDM) in rats during catabolic response of fracture healing. There were conflicting results regarding the efficacy of PBM on bone healing process in healthy and diabetic animals. Forty-eight rats have been distributed into four groups: group 1 (healthy control, no TIDM and no PBM), group 2 (healthy test, no TIDM and PBM), group 3 (diabetic control, TIDM and no PBM), and group 4 (diabetic test, no TIDM and PBM). TIDM was induced in the groups 3 and 4. A partial bone defect in tibia was made in all groups. The bone defects of groups second and fourth were irradiated by a laser (890 nm, 80 Hz, 1.5 J/cm 2 ). Thirty days after the surgery, all bone defects were extracted and were submitted to stereological examination and real-time polymerase chain reaction (RT-PCR). PBM significantly increased volumes of total callus, total bone, bone marrow, trabecular bone, and cortical bone, and the numbers of osteocytes and osteoblasts of callus in TIDM rats compared to those of callus in diabetic control. In addition, TIDM increased RUNX2, and osteocalcin in callus of tibial bone defect compared to healthy group. PBM significantly decreased osteocalcin gene expression in TIDM rats. PBM significantly increased many stereological parameters of bone repair in an STZ-induced TIDM during catabolic response of fracture healing. Further RT-PCR test demonstrated that bone repair was modulated in diabetic rats during catabolic response of fracture healing by significant increase in mRNA expression of RUNX2, and osteocalcin compared to healthy control rats. PBM also decreased osteocalcin mRNA expression in TIDM rats.

Development of In Vitro Systems for Switchgrass (Panicum virgatum) - Final Report for 1992 to 2002

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conger, B.V.

2003-01-16

Our project began on July 1, 1992, with the objective of developing systems that could be used in biotechnological approaches to switchgrass improvement. Within six months after initiation of the project, we had worked out protocols in which plants could be regenerated from callus cultures through both organogenesis and somatic embryogenesis. Documentation for both modes of regeneration was provided in our progress reports and in publications. One thousand regenerated plants were established in the field during the first year. We found that Alamo (lowland type) was much more amenable to in vitro culture, and plants could be regenerated much moremore » easily than from Cave-in-Rock (upland type). During the first three years of the project, we studied the influence of genotype, culture medium components, explant type, etc., on regeneration. As mentioned, we found that the lowland cultivars Alamo and Kanlow were much easier to regenerate than upland cultivars, such as Trailblazer, Blackwell, and Cave-in-Rock. For callus induction, we initially used mature caryopses, young leaf tissue, and portions of seedlings. We were successful in inducing callus and regenerating plants from all explants. Two other systems developed during the 4th to 6th year period of the project included multiple shoot formation initiated from germinated seedlings and regenerable suspension cultures. The latter were initiated from embryogenic calluses produced from in vitro developed inflorescences. An important factor for producing multiple shoots was the presence of thidiazuron in the medium. The shoots could be easily rooted and numerous plantlets produced. The last 3 to 4 years of the project focused on anther and microspore culture experiments to produce haploid plants and on genetic transformation. Although thousands of putative haploid plants were produced from a few anthers, they were very weak and difficult to keep alive. Chromosome counts revealed the gametic number in cells where it was possible to count chromosomes. The isolated microspore culture experiments were not successful.« less

Targeted delivery of lovastatin and tocotrienol to fracture site promotes fracture healing in osteoporosis model: micro-computed tomography and biomechanical evaluation.

PubMed

Ibrahim, Nurul 'Izzah; Khamis, Mohd Fadhli; Mod Yunoh, Mohd Faridz; Abdullah, Shahrum; Mohamed, Norazlina; Shuid, Ahmad Nazrun

2014-01-01

Osteoporosis is becoming a major health problem that is associated with increased fracture risk. Previous studies have shown that osteoporosis could delay fracture healing. Although there are potential agents available to promote fracture healing of osteoporotic bone such as statins and tocotrienol, studies on direct delivery of these agents to the fracture site are limited. This study was designed to investigate the effects of two potential agents, lovastatin and tocotrienol using targeted drug delivery system on fracture healing of postmenopausal osteoporosis rats. The fracture healing was evaluated using micro CT and biomechanical parameters. Forty-eight Sprague-Dawley female rats were divided into 6 groups. The first group was sham-operated (SO), while the others were ovariectomized (OVx). After two months, the right tibiae of all rats were fractured at metaphysis region using pulsed ultrasound and were fixed with plates and screws. The SO and OVxC groups were given two single injections of lovastatin and tocotrienol carriers. The estrogen group (OVx+EST) was given daily oral gavages of Premarin (64.5 µg/kg). The Lovastatin treatment group (OVx+Lov) was given a single injection of 750 µg/kg lovastatin particles. The tocotrienol group (OVx+TT) was given a single injection of 60 mg/kg tocotrienol particles. The combination treatment group (OVx+Lov+TT) was given two single injections of 750 µg/kg lovastatin particles and 60 mg/kg tocotrienol particles. After 4 weeks of treatment, the fractured tibiae were dissected out for micro-CT and biomechanical assessments. The combined treatment group (OVx+Lov+TT) showed significantly higher callus volume and callus strength than the OVxC group (p<0.05). Both the OVx+Lov and OVx+TT groups showed significantly higher callus strength than the OVxC group (p<0.05), but not for callus volume. In conclusion, combined lovastatin and tocotrienol may promote better fracture healing of osteoporotic bone.

Production and optimisation of rosmarinic acid by Satureja hortensis L. callus cultures.

PubMed

Tepe, Bektas; Sokmen, Atalay

2007-11-01

In this study, production and optimisation of rosmarinic acid, a phenolic acid and an economically important metabolite, was investigated in the callus cultures established from the mature seeds of Satureja hortensis L. (summer savory) plant. Gamborg's B5 basal medium, supplemented with indol butyric acid (IBA) (1.00 mg L(-1)), N6-benzyl aminopurine (6-BA) (1.00 mg L(-1)) and sucrose (2.5%, w/v), was employed for the establishment and maintenance of the callus cultures. Applications were individually prepared by preparing the media containing different IBA/6-BA combinations and sucrose concentrations. All of the applications were carried out in the continuous dark. In the applications, where the effects of IBA/6-BA combinations on the growth and rosmarinic acid accumulation were assayed (1-15 applications), the highest biomass yield was obtained from the medium supplemented with 1.00 mg L(-1) IBA and 5.00 mg L(-1) 6-BA. In the case of the rosmarinic acid accumulation, an opposite relationship was determined between the growth and rosmarinic acid production. While the highest biomass yield was obtained from the medium containing 1.00 mg L(-1) IBA and 5.00 mg L(-1) 6-BA, the highest rosmarinic acid accumulation was obtained from the medium supported with 1.00 mg L(-1) IBA and 1.00 mg L(-1) 6-BA. In the applications where the effects of sucrose concentrations on the growth and rosmarinic acid accumulation were examined, the highest biomass yield was obtained from the medium which is supplemented with 5.0% (w/v) sucrose. In this category, the highest rosmarinic acid accumulation was obtained from the medium which is supported with 3.0% (w/v) sucrose. According to the experiments carried out with the wild S. hortensis, it is found to have 25.02+/-1.21 mg g(-1) rosmarinic acid. No differentiation was observed in any callus during the course of this study.

Short-term muscle atrophy caused by botulinum toxin-A local injection impairs fracture healing in the rat femur.

PubMed

Hao, Yongqiang; Ma, Yongcheng; Wang, Xuepeng; Jin, Fangchun; Ge, Shengfang

2012-04-01

Damaged bone is sensitive to mechanical stimulation throughout the remodeling phase of bone healing. Muscle damage and muscular atrophy associated with open fractures and subsequent fixation are not beneficial to maintaining optimum conditions for mechanical stability. The aim of this study was to investigate whether local muscle atrophy and dysfunction affect fracture healing in a rat femur fracture model. We combined the rat model of a short period atrophy of the quadriceps with femur fracture. Forty-four-month-old male Wistar rats were adopted for this study. Two units of botulinum toxin-A (BXTA) were administered locally into the right side of the quadriceps of each rat, while the same dose of saline was injected into the contralateral quadriceps. After BXTA had been fully absorbed by the quadriceps, osteotomy was performed in both femurs with intramedullary fixation. Gross observation and weighing of muscle tissue, X-ray analysis, callus histology, and bone biomechanical testing were performed at different time points up to 8 weeks post-surgery. Local injection of BXTA led to a significant decrease in the volume and weight of the quadriceps compared to the control side. At the eighth week, the left side femurs of the saline-injected quadriceps almost reached bony union, and fibrous calluses were completely calcified into woven bone. However, a gap was still visible in the BXTA-treated side on X-ray images. As showed by bone histology, there were no mature osseous calluses or woven bone on the BXTA-treated side, but a resorption pattern was evident. Biomechanical testing indicated that the femurs of the BXTA-treated side exhibited inferior mechanical properties compared with the control side. The inferior outcome following BXTA injection, compared with saline injection, in terms of callus resistance may be the consequence of unexpected load and mechanical unsteadiness caused by muscle atrophy and dysfunction. Copyright © 2011 Orthopaedic Research Society.

[Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

PubMed

Wei, Yue-Rong; Huang, Xue-Lin; Li, Jia; Huang, Xia; Li, Zhe; Li, Xiao-Ju

2005-01-01

Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV ECS/30 mL medium in 100 mL flask, and the appropriate subculture cycle was 15 days. Planting of 6 months old ECS on semi-solid medium of somatic embryo induction and development (MSD) resulted in approximately 280 x 10(3) somatic embryos/mL PCV ECS. MSD contained SH macronutrients, micro-nutrients, Fe-EDTA and MS vitamins supplemented with 4.5 micromol/L biotin, 680 micromol/L glutamine, 2 mmol/L proline, 100 mg/L malt extract, 1.1 micromol/L NAA, 0.2 micromol/L zeatin, 0.5 micromol/L kinetin, 0.7 micromol/L N6-(2-isopentenyl) adenine, 29 mmol/L lactose, 130 mmol/L sucrose and solidified with 2g/L gelrite. After 3 months of maturity on MSD, 17.28% of the somatic embryos were germinated on germination media (MG), consisted of MS salt, Morel and Wetmore vitamins, 0.2 micromol/L 6-BA, 1.1 micromol/L IAA, 87 micromol/L sucrose and solidified with 2 g/L gelrite; and 14.16% of the somatic embryos could develop into normal plantlets on rooting media contained the same composition as that of MG but without auxin and cytokinin.

Plantlet regeneration potential from seedling explants of vitegnus (Vitex agnus castus).

PubMed

Chamandoosti, F

2007-11-15

In this research a simple and repeatable method for regeneration of a important medicinal plant (Vitex agnus castus) described. Different seedling explants such as hypocotyl, cotyledon, root and apical meristem were cultured in MS basal media with different kinds and concentrations of PGRs. Root and apical meristem explants were the only explants that have regeneration whole plantlets potential. It was interesting that regeneration whole plantlets from root and apical meristem explants have different developmental pathways. Whole plantlets from apical meristem explants regenerated by passing phase callusing whereas regeneration whole plantlets from root was direct and without phase callusing. This subject implies that we can have many manipulation possibilities in order to different objects of tissue culture by selecting different explants in vitegnus.

Hemp (Cannabis sativa L.).

PubMed

Feeney, Mistianne; Punja, Zamir K

2015-01-01

Hemp (Cannabis sativa L.) suspension culture cells were transformed with Agrobacterium tumefaciens strain EHA101 carrying the binary plasmid pNOV3635. The plasmid contains a phosphomannose isomerase (PMI) selectable marker gene. Cells transformed with PMI are capable of metabolizing the selective agent mannose, whereas cells not expressing the gene are incapable of using the carbon source and will stop growing. Callus masses proliferating on selection medium were screened for PMI expression using a chlorophenol red assay. Genomic DNA was extracted from putatively transformed callus lines, and the presence of the PMI gene was confirmed using PCR and Southern hybridization. Using this method, an average transformation frequency of 31.23% ± 0.14 was obtained for all transformation experiments, with a range of 15.1-55.3%.

Papaya (Carica papaya L.).

PubMed

Zhu, Yun J; Fitch, Maureen M M; Moore, Paul H

2006-01-01

Transgenic papaya plants were initially obtained using particle bombardment, a method having poor efficiency in producing intact, single-copy insertion of transgenes. Single-copy gene insertion was improved using Agrobacterium tumefaciens. With progress being made in genome sequencing and gene discovery, there is a need for more efficient methods of transformation in order to study the function of these genes. We describe a protocol for Agrobacterium-mediated transformation using carborundum-wounded papaya embryogenic calli. This method should lead to high-throughput transformation, which on average produced at least one plant that was positive in polymerase chain reaction (PCR), histochemical staining, or by Southern blot hybridization from 10 to 20% of the callus clusters that had been co-cultivated with Agrobacterium. Plants regenerated from the callus clusters in 9 to 13 mo.

Pineapple [Ananas comosus (L.) Merr].

PubMed

Yabor, Lourdes; Espinosa, Patricia; Arencibia, Ariel D; Lorenzo, José C

2006-01-01

A procedure for pineapple [Ananas comosus (L.) Merr.] genetic transformation is described, which involves temporary immersion bioreactors (TIB) for selection of transgenic plants. Success in the production of transgenic pineapple plants combines tissue culture factors. Firstly, the use of regenerable pineapple callus as starting material for transformation whose cells shown to be competent for Agrobacterium infection. Secondly, the used of filtered callus, resulting in homogeneously sized clusters, thereby increasing the contact between the cell surfaces and A. tumefaciens and releasing phenolic compounds which induce Agrobacterium virulence. Thirdly, regeneration of primary plants without selection pressure, that allowing a massive production of putative transgenic pineapples. Finally, we support that TIB technology is a powerful system to recover nonchimera transgenic plants by micropropagation with the use of an adequate selection agent.

Online structural elucidation of alkaloids and other constituents in crude extracts and cultured cells of Nandina domestica by combination of LC-MS/MS, LC-NMR, and LC-CD analyses.

PubMed

Iwasa, Kinuko; Takahashi, Teturo; Nishiyama, Yumi; Moriyasu, Masataka; Sugiura, Makiko; Takeuchi, Atsuko; Tode, Chisato; Tokuda, Harukuni; Takeda, Kazuyoshi

2008-08-01

The combination of NMR, MS, and CD data permitted the structural elucidation including the absolute configuration of the known alkaloids and unknown components in the extract matrix solution of Nandina domestica without isolation and sample purification prior to the coupling experiments. Unstable natural stereoisomers were identified by LC-NMR and LC-MS. Five known alkaloids, (S)-isoboldine, (S)-domesticine, (S)-nantenine, sinoacutine, and menispermine, were identified from N. domestica. O-Methylpallidine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were also characterized for the first time from this plant. Known jatrorrhizine, palmatine, and berberine and unknown (R)-carnegine and (E, E)-, (E, Z)-, and (Z, Z)-terrestribisamide were identified in the callus of N. domestica.

Multicellular structures developing during maize microspore culture express endosperm and embryo-specific genes and show different embryogenic potentialities.

PubMed

Massonneau, Agnes; Coronado, Maria-José; Audran, Arthur; Bagniewska, Agnieszka; Mòl, Rafal; Testillano, Pilar S; Goralski, Grzegorz; Dumas, Christian; Risueño, Maria-Carmen; Matthys-Rochon, Elisabeth

2005-07-01

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.

Responses of meristematic callus cells of two Cynodon dactylon genotypes to aluminium.

PubMed

Ramgareeb, Sumita; Cooke, John A; Watt, M Paula

2004-11-01

Responses to Al3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al3+ toxicity and resistance. A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al3+ ion in culture media, which was supplied as 0.08-2.3 mM Al3+ for 2-8 weeks. Increasing Al3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells. Exposure to 0.8 mM Al3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected. In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis. The Al-R cells appeared to exclude Al3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R). The results showed that by studying the cellular responses to Al3+ it is possible to discriminate between the Al-S and Al-R C. dactylon genotypes.

Antioxidant Activity and Total Phenolic and Flavonoid Content of Various Solvent Extracts from In Vivo and In Vitro Grown Trifolium pratense L. (Red Clover)

PubMed Central

Mat Taha, Rosna; Banisalam, Behrooz

2015-01-01

In the present study the extracts of in vivo and in vitro grown plants as well as callus tissue of red clover were tested for their antioxidant activities, using different extraction solvent and different antioxidant assays. The total flavonoid and phenolic contents as well as extraction yield of the extracts were also investigated to determine their correlation with the antioxidant activity of the extracts. Among all the tested extracts the highest amounts of total phenolic and total flavonoids content were found in methanol extract of in vivo grown plants. The antioxidant activity of tested samples followed the order in vivo plant extract > callus extract > in vitro extract. The highest reducing power, 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging, and chelating power were found in methanol extracts of in vivo grown red clover, while the chloroform fraction of in vivo grown plants showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, superoxide anion radical scavenging and hydrogen peroxide scavenging compared to the other tested extracts. A significant correlation was found between the antioxidant activity of extracts and their total phenolic and total flavonoid content. According to the findings, the extract of in vitro culture of red clover especially the callus tissue possesses a comparable antioxidant activity to the in vivo cultured plants' extract. PMID:26064936

WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis[OPEN

PubMed Central

Ohnuma, Mariko; Kurata, Tetsuya; Nakata, Masaru; Ohme-Takagi, Masaru

2017-01-01

Many plant species display remarkable developmental plasticity and regenerate new organs after injury. Local signals produced by wounding are thought to trigger organ regeneration but molecular mechanisms underlying this control remain largely unknown. We previously identified an AP2/ERF transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1) as a central regulator of wound-induced cellular reprogramming in plants. In this study, we demonstrate that WIND1 promotes callus formation and shoot regeneration by upregulating the expression of the ENHANCER OF SHOOT REGENERATION1 (ESR1) gene, which encodes another AP2/ERF transcription factor in Arabidopsis thaliana. The esr1 mutants are defective in callus formation and shoot regeneration; conversely, its overexpression promotes both of these processes, indicating that ESR1 functions as a critical driver of cellular reprogramming. Our data show that WIND1 directly binds the vascular system-specific and wound-responsive cis-element-like motifs within the ESR1 promoter and activates its expression. The expression of ESR1 is strongly reduced in WIND1-SRDX dominant repressors, and ectopic overexpression of ESR1 bypasses defects in callus formation and shoot regeneration in WIND1-SRDX plants, supporting the notion that ESR1 acts downstream of WIND1. Together, our findings uncover a key molecular pathway that links wound signaling to shoot regeneration in plants. PMID:28011694

Production of herbicide-resistant transgenic Panax ginseng through the introduction of the phosphinothricin acetyl transferase gene and successful soil transfer.

PubMed

Choi, Y E; Jeong, J H; In, J K; Yang, D C

2003-02-01

Herbicide-resistant transgenic Panax ginseng plants were produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through Agrobacterium tumefaciens co-cultivation. Embryogenic callus gathered from cotyledon explants of P. ginseng were pre-treated with 0.5 M sucrose or 0.05 M MgSO(4 )before Agrobacterium infection. This pre-treatment process markedly enhanced the transient expression of the beta-glucuronidase (GUS) gene. Embryogenic callus was initially cultured on MS medium supplemented with 400 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 25 mg/l kanamycin and 300 mg/l cefotaxime. Somatic embryos formed on the surfaces of kanamycin-resistant callus. Upon development into the cotyledonary stage, these somatic embryos were transferred to a medium containing 50 mg/l kanamycin and 5 mg/l gibberellic acid to induce germination and strong selection. Integration of the transgene into the plants was confirmed by polymerase chain reaction and Southern analyses. Transfer of the transgenic ginseng plantlets to soil was successfully accomplished via acclimatization in autoclaved perlite. Not all of the plantlets survived in soil that had not been autoclaved because of fungal infection, particularly in the region between the roots and leaves. Transgenic plants growing in soil were observed to be strongly resistant to Basta application.

Dynamic Stabilization of Simple Fractures With Active Plates Delivers Stronger Healing Than Conventional Compression Plating

PubMed Central

Tsai, Stanley; Bliven, Emily K.; von Rechenberg, Brigitte; Kindt, Philipp; Augat, Peter; Henschel, Julia; Fitzpatrick, Daniel C.; Madey, Steven M.

2017-01-01

Objectives: Active plates dynamize a fracture by elastic suspension of screw holes within the plate. We hypothesized that dynamic stabilization with active plates delivers stronger healing relative to standard compression plating. Methods: Twelve sheep were randomized to receive either a standard compression plate (CP) or an active plate (ACTIVE) for stabilization of an anatomically reduced tibial osteotomy. In the CP group, absolute stabilization was pursued by interfragmentary compression with 6 cortical screws. In the ACTIVE group, dynamic stabilization after bony apposition was achieved with 6 elastically suspended locking screws. Fracture healing was analyzed weekly on radiographs. After sacrifice 9 weeks postsurgery, the torsional strength of healed tibiae and contralateral tibiae was measured. Finally, computed tomography was used to assess fracture patterns and healing modes. Results: Healing in both groups included periosteal callus formation. ACTIVE specimens had almost 6 times more callus area by week 9 (P < 0.001) than CP specimens. ACTIVE specimens recovered on average 64% of their native strength by week 9, and were over twice as strong as CP specimens, which recovered 24% of their native strength (P = 0.008). Microcomputed tomography demonstrated that compression plating induced a combination of primary bone healing and gap healing. Active plating consistently stimulated biological bone healing by periosteal callus formation. Conclusions: Compared with compression plating, dynamic stabilization of simple fractures with active plates delivers significantly stronger healing. PMID:27861456

Quantification of Anti-Addictive Alkaloids Ibogaine and Voacangine in In Vivo- and In Vitro-Grown Plants of Two Mexican Tabernaemontana Species.

PubMed

Krengel, Felix; Herrera Santoyo, Josefina; Olivera Flores, Teresa de Jesús; Chávez Ávila, Víctor M; Pérez Flores, Francisco J; Reyes Chilpa, Ricardo

2016-12-01

Tabernaemontana alba and Tabernaemontana arborea are Apocynaceae species used in Mexican traditional medicine for which little phytochemical information exists. In this study, preliminary gas chromatography/mass spectrometry analyses of different organs obtained from wild plants of both species identified a total of 10 monoterpenoid indole alkaloids (MIAs) and one simple indole alkaloid, nine of which were reported for the first time in these species. Furthermore, callus cultures were established from T. alba leaf explants and regeneration of whole plants was accomplished via somatic embryogenesis. The anti-addictive MIAs ibogaine and voacangine were then quantified by gas chromatography with flame ionization detection in wild plants of both species, as well as greenhouse-grown plants, in vitro-grown plantlets and embryogenic callus of T. alba. Ibogaine and voacangine were present in most samples taken from the whole plants of both species, with stem and root barks showing the highest concentrations. No alkaloids were detected in callus samples. It was concluded that T. alba and T. arborea are potentially viable sources of ibogaine and voacangine, and that these MIAs can be produced through somatic embryogenesis and whole plant regeneration of T. alba. Approaches to increase MIA yields in whole plants and to achieve alkaloid production directly in cell cultures are discussed. © 2016 Wiley-VHCA AG, Zurich, Switzerland.

Bulimia

MedlinePlus

... large amounts of high-calorie foods, usually in secret. During these episodes, the person feels a lack ... and pimples Small cuts and calluses across the tops of the finger joints from forcing oneself to ...

Applications of optical manipulation in plant biology

NASA Astrophysics Data System (ADS)

Buer, Charles S.

Measuring small forces in biology is important for determining basic physiological parameters of a cell. The plant cell wall provides a primary defense and presents a barrier to research. Magnitudes of small forces are impossible to measure with mechanical transducers, glass needles, atomic force microscopy, or micropipet-based force transduction due to the cell wall. Therefore, a noninvasive method of breaching the plant cell wall to access the symplastic region of the cell is required. Laser light provides sub-micrometer positioning, particle manipulation without mechanical contact, and piconewton force determination. Consequently, the extension of laser microsurgery to expand an experimental tool for plant biology encompassed the overall objective. A protocol was developed for precisely inserting microscopic objects into the periplasmic region of plant callus cells using laser microsurgery. Ginkgo biloba and Agrobacterium rhizogenes were used as the model system for developing the optical tweezers and scalpel techniques. Better than 95% survival was achieved after plasmolyzing G. biloba cells, ablating a 2-4 μm hole through the cell wall using a pulsed UV laser beam, trapping and manipulating bacteria into the periplasmic region, and deplasmolyzing the cells. Optical trapping experiments implied a difference existed between the bacteria models. Determining the optical trapping efficiency of Agrobacterium rhizogenes and A. tumefaciens strains indicated the A. rhizogenes strain, ATCC 11325, was significantly less efficiently trapped than strains A4 and ATCC 15834 and the A. tumefaciens strain LBA4404. Differences were also found in capsule generation, growth media viscosity, and transmission electron microscopy negative staining implying that a difference in surface structure exists. Calcofluor fluorescence suggests the difference involves an exopolysaccharide. Callus cell plasmolysis revealed Hechtian strands interconnecting the plasma membrane and the cell wall. The spring tension of these strands was measured in normal and cold-hardened G. biloba and N. tabacum callus cells. There was little change in flexibility between the groups of cultured cells in either species studied. Microspheres were attached to Hechtian strands in normal cultured Nicotiana tabacum and the cells were deplasmolyzed and replasmolyzed to determine the fate of Hechtian strands. The microspheres either moved to the plasma membrane and adhered or moved to the cell wall and adhered. The attached microspheres occasionally moved independently on the same strand. Inserted microspheres provided a visual probe to follow physiological events within a plant cell.

Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos.

PubMed

Belide, Srinivas; Vanhercke, Thomas; Petrie, James Robertson; Singh, Surinder Pal

2017-01-01

Sorghum ( Sorghum bicolor L.) is one of the world's most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency. We report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5-2.0 mm in length) isolated 12-15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt - II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs. This protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.

Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

PubMed

Mamedes-Rodrigues, T C; Batista, D S; Vieira, N M; Matos, E M; Fernandes, D; Nunes-Nesi, A; Cruz, C D; Viccini, L F; Nogueira, F T S; Otoni, W C

2018-03-01

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L -1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

16 CFR 18.0 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... greenhouse plants solely for inside culture or use and annual vegetable plants. Industry members. Any person... seeds, cuttings, callus or other plant tissue, spores or other propagules under a controlled environment...

16 CFR 18.0 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... greenhouse plants solely for inside culture or use and annual vegetable plants. Industry members. Any person... seeds, cuttings, callus or other plant tissue, spores or other propagules under a controlled environment...

16 CFR 18.0 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... greenhouse plants solely for inside culture or use and annual vegetable plants. Industry members. Any person... seeds, cuttings, callus or other plant tissue, spores or other propagules under a controlled environment...

Arsenic speciation for the phytoremediation by the Chinese brake fern, Pteris vittata.

PubMed

Shoji, R; Yajima, R; Yano, Y

2008-01-01

Arsenic (As) speciation for the phytoremediation by the Chinese brake fern was studied. In particular, the mechanism of how plants induce compounds containing thiol (SH) and proteins by As exposure in terms of the relationship between As and phosphate uptaken into plant cells was examined. Pteris vittata callus could efficiently reduce As(V) to As(III) by the rapid introduction of reductase and synthesize thiols leading to phytochelatins production. Furthermore, Pteris vittata could control phosphate concentration in the cells corresponding to the concentration of arsenite and arsenate. To our best knowledge, this is the first report to show the mechanisms of such high As tolerance of Pteris vittata using their callus in terms of in vitro approach for the analysis of As speciation and metabolism route.

First report of biomimetic synthesis of silver nanoparticles using aqueous callus extract of Centella asiatica and their antimicrobial activity

NASA Astrophysics Data System (ADS)

Netala, Vasudeva Reddy; Kotakadi, Venkata Subbaiah; Nagam, Venkateswarlu; Bobbu, Pushpalatha; Ghosh, Sukhendu Bikash; Tartte, Vijaya

2015-10-01

The present study reports the simple and eco-friendly approach for biosynthesis of silver nanoparticles (AgNPs) using aqueous callus extract as reducing agent for the first time. The formation of AgNPs was initially confirmed by characteristic surface plasmon resonance (SPR) peak 453 nm by UV-Visible spectroscopy. FTIR spectrum shows different functional groups which probably involved in the synthesis and stabilization of AgNPs. TEM analysis determined the well-dispersed AgNPs with roughly spherical shape and size ranging 5-40 nm. XRD patterns revealed the crystalline nature of AgNPs with face-centered cubic (fcc) lattice. The synthesized AgNPs were found to have strong inhibitory activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa.

Microcalli Induction in Protoplasts Isolated from Embryogenic Callus of Date Palm.

PubMed

Titouh, Khayreddine; Boufis, Nazim; Khelifi, Lakhdar

2017-01-01

Date palm (Phoenix dactylifera L.) production is severely hampered due to several pests and diseases. Biotechnological tools such as protoplast fusion appear as an alternative to ensure rapid genetic improvement and multiplication of this species. However, establishment of an effective system of plant regeneration from protoplasts culture is a prerequisite for date palm somatic hybridization. In this chapter, we describe an effective protocol to induce microcalli in protoplasts isolated from nodular callus of important Algerian date palm cultivars. In this protocol, the main factors influencing the isolation (i.e., enzymatic solution, mannitol concentration, duration, and mode of maceration) of protoplasts from the calli of Algerian date palm cultivars were optimized. Purified protoplasts were cultured on a semisolid medium supplemented with a hormonal balance of auxin and cytokinin to obtain microcalli formation.

Biomechanics of bone-fracture fixation by stiffness-graded plates in comparison with stainless-steel plates

PubMed Central

Ganesh, VK; Ramakrishna, K; Ghista, Dhanjoo N

2005-01-01

Background In the internal fixation of fractured bone by means of bone-plates fastened to the bone on its tensile surface, an on-going concern has been the excessive stress-shielding of the bone by the excessively-stiff stainless-steel plate. The compressive stress-shielding at the fracture-interface immediately after fracture-fixation delays callus formation and bone healing. Likewise, the tensile stress-shielding of the layer of the bone underneath the plate can cause osteoporosis and decrease in tensile strength of this layer. Method In order to address this problem, we propose to use stiffness-graded plates. Accordingly, we have computed (by finite-element analysis) the stress distribution in the fractured bone fixed by composite plates, whose stiffness is graded both longitudinally and transversely. Results It can be seen that the stiffness-graded composite-plates cause less stress-shielding (as an example: at 50% of the healing stage, stress at the fracture interface is compressive in nature i.e. 0.002 GPa for stainless steel plate whereas stiffness graded plates provides tensile stress of 0.002 GPa. This means that stiffness graded plate is allowing the 50% healed bone to participate in loadings). Stiffness-graded plates are more flexible, and hence permit more bending of the fractured bone. This results in higher compressive stresses induced at the fractured faces accelerate bone-healing. On the other hand, away from the fracture interface the reduced stiffness and elastic modulus of the plate causes the neutral axis of the composite structure to be lowered into the bone resulting in the higher tensile stress in the bone-layer underneath the plate, wherein is conducive to the bone preserving its tensile strength. Conclusion Stiffness graded plates (with in-built variable stiffness) are deemed to offer less stress-shielding to the bone, providing higher compressive stress at the fractured interface (to induce accelerated healing) as well as higher tensile stress in the intact portion of the bone (to prevent bone remodeling and osteoporosis). PMID:16045807

Diabetes and Foot Problems

MedlinePlus

... medicated pads use liquid corn and callus removers Cutting and over-the counter corn removal products can ... nonsharp nail file. Trimming this way helps prevent cutting your skin and keeps the nails from growing ...

Low Intensity Pulsed Ultrasound Enhanced Mesenchymal Stem Cell Recruitment through Stromal Derived Factor-1 Signaling in Fracture Healing

PubMed Central

Wei, Fang-Yuan; Leung, Kwok-Sui; Li, Gang; Qin, Jianghui; Chow, Simon Kwoon-Ho; Huang, Shuo; Sun, Ming-Hui; Qin, Ling; Cheung, Wing-Hoi

2014-01-01

Low intensity pulsed ultrasound (LIPUS) has been proven effective in promoting fracture healing but the underlying mechanisms are not fully depicted. We examined the effect of LIPUS on the recruitment of mesenchymal stem cells (MSCs) and the pivotal role of stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) pathway in response to LIPUS stimulation, which are essential factors in bone fracture healing. For in vitro study, isolated rat MSCs were divided into control or LIPUS group. LIPUS treatment was given 20 minutes/day at 37°C for 3 days. Control group received sham LIPUS treatment. After treatment, intracellular CXCR4 mRNA, SDF-1 mRNA and secreted SDF-1 protein levels were quantified, and MSCs migration was evaluated with or without blocking SDF-1/CXCR4 pathway by AMD3100. For in vivo study, fractured 8-week-old young rats received intracardiac administration of MSCs were assigned to LIPUS treatment, LIPUS+AMD3100 treatment or vehicle control group. The migration of transplanted MSC to the fracture site was investigated by ex vivo fluorescent imaging. SDF-1 protein levels at fracture site and in serum were examined. Fracture healing parameters, including callus morphology, micro-architecture of the callus and biomechanical properties of the healing bone were investigated. The in vitro results showed that LIPUS upregulated SDF-1 and CXCR4 expressions in MSCs, and elevated SDF-1 protein level in the conditioned medium. MSCs migration was promoted by LIPUS and partially inhibited by AMD3100. In vivo study demonstrated that LIPUS promoted MSCs migration to the fracture site, which was associated with an increase of local and serum SDF-1 level, the changes in callus formation, and the improvement of callus microarchitecture and mechanical properties; whereas the blockade of SDF-1/CXCR4 signaling attenuated the LIPUS effects on the fractured bones. These results suggested SDF-1 mediated MSCs migration might be one of the crucial mechanisms through which LIPUS exerted influence on fracture healing. PMID:25181476

High dose teriparatide (rPTH1-34) therapy increases callus volume and enhances radiographic healing at 8-weeks in a massive canine femoral allograft model.

PubMed

Nishitani, Kohei; Mietus, Zachary; Beck, Christopher A; Ito, Hiromu; Matsuda, Shuichi; Awad, Hani A; Ehrhart, Nicole; Schwarz, Edward M

2017-01-01

Small animal studies have demonstrated significant high-dose recombinant parathyroid hormone1-34 (rPTH1-34) effects on intercalary allograft healing. Towards a human adjuvant therapy to decrease non-unions, we evaluated rPTH1-34 safety and efficacy in a clinically relevant canine femoral allograft model. Adult female mongrel hounds (n = 20) received a 5cm mid-diaphyseal osteotomy reconstructed with a plated allograft, and were randomized to: 1) Placebo (n = 5; daily saline), 2) Continuous rPTH1-34 (n = 7; 5 μg/kg/day s.c. from day 1-55 post-op), or 3) Delayed rPTH1-34 (n = 8; 5 μg/kg/day s.c. from day 14-28 post-op). Safety was assessed by physical behavior and blood calcium monitoring. Cone beam CT (CB-CT) was performed on days 14, 28 and 56 post-op to assess 2D cortical healing, 3D bone volume, and Union Ratio. Biomechanical testing and dynamic histomorphometry were also performed. The high drug dose was poorly tolerated, as most dogs receiving rPTH1-34 had to be given intravenous saline, and one dog died from hypercalcemia. Continuous rPTH1-34 significantly increased 2D healing and callus volumes at 4-weeks versus Placebo, and sustained the significant increase in cortical union at 8-week (p<0.05). These rPTH1-34 effects were confirmed by histomorphometry, revealing significant increases in mineral apposition rates (MAR) on host bone and graft-host junctions (p<0.05). Delayed rPTH1-34 significantly increased callus volume and MAR at 8 weeks (p<0.05). Although no biomechanical differences were observed, as expected for early healing, the results demonstrated that 2D RUST scoring significantly correlated with torsional biomechanics (p<0.01). In conclusion, 8-weeks of intermittent high-dose rPTH1-34 treatment significantly increases callus formation and accelerates bony union of intercalary massive allografts in a clinically relevant canine model, but with serious side-effects from hypercalcemia.

Rrhizogenesis in vitro is a convenient model for studying the root graviperceptive apparatus formation in microgravity

NASA Astrophysics Data System (ADS)

Kordyum, Elizabeth; Sarnatska, Veresa; Ovcharenko, Yulia

A root graviperceptive apparatus is known to form in microgravity but does not function in the absence of a gravitational vector, that has been shown in many spaceflight experiments with seedlings of different plant species. In statocytes, which are differentiated in microgravity, a nucleus is localized in the proximal part of a cell as at 1 g. Unlike control, amyloplastsstatoliths do not sedimented in the distal part of a cell in microgravity, they group in the cell center more often, sometimes they localized in the different part of a cell. In all these experiments, the objects of investigations were embryonal roots formed in seeds at 1 g. There is only single report that columella cells in roots, which developed de novo from callus in space flight, did not differentiate in statocytes. Therefore, we call to attention to rhizogenesis in vitro as a convenient model for studying the influence of microgravity on differentiation of a root graviperceptive apparatus. Two methods for obtaining of Arabidopsis thaliana roots in vitro are proposed: the first-from the primary callus of leaf origin and the second - from leaf fragments. Callus initiation and growth are successful on MS medium supplemented with vitamin B5, glycine, inositol, 2,4-D, kinetin, glucose and agar. For induction of rhizogenesis calli were transferred to medium without hormones or medium which contained one to ten of MS mineral salts and microelements, without vitamins and hormones. Rhyzogenesis was obtained without added growth substances, but considerably higher number of calli with roots and number of roots per callus are on MS medium diluted tenfold. Rhizogenesis in A. thaliana leaf segments should present no problem, but the most intensive root formation is obtained when culturing them for three day on diluted MS medium supplemented with salycilic acid and then on diluted MS medium only. The low temperature treatment for three days increases the number of roots formed. A role of both plasticity and positional keys in vivo and in vitro root development at 1 g and under clinorotation is discussed.

High dose teriparatide (rPTH1-34) therapy increases callus volume and enhances radiographic healing at 8-weeks in a massive canine femoral allograft model

PubMed Central

Mietus, Zachary; Beck, Christopher A.; Ito, Hiromu; Matsuda, Shuichi; Awad, Hani A.; Ehrhart, Nicole; Schwarz, Edward M.

2017-01-01

Small animal studies have demonstrated significant high-dose recombinant parathyroid hormone1-34 (rPTH1-34) effects on intercalary allograft healing. Towards a human adjuvant therapy to decrease non-unions, we evaluated rPTH1-34 safety and efficacy in a clinically relevant canine femoral allograft model. Adult female mongrel hounds (n = 20) received a 5cm mid-diaphyseal osteotomy reconstructed with a plated allograft, and were randomized to: 1) Placebo (n = 5; daily saline), 2) Continuous rPTH1-34 (n = 7; 5 μg/kg/day s.c. from day 1–55 post-op), or 3) Delayed rPTH1-34 (n = 8; 5 μg/kg/day s.c. from day 14–28 post-op). Safety was assessed by physical behavior and blood calcium monitoring. Cone beam CT (CB-CT) was performed on days 14, 28 and 56 post-op to assess 2D cortical healing, 3D bone volume, and Union Ratio. Biomechanical testing and dynamic histomorphometry were also performed. The high drug dose was poorly tolerated, as most dogs receiving rPTH1-34 had to be given intravenous saline, and one dog died from hypercalcemia. Continuous rPTH1-34 significantly increased 2D healing and callus volumes at 4-weeks versus Placebo, and sustained the significant increase in cortical union at 8-week (p<0.05). These rPTH1-34 effects were confirmed by histomorphometry, revealing significant increases in mineral apposition rates (MAR) on host bone and graft-host junctions (p<0.05). Delayed rPTH1-34 significantly increased callus volume and MAR at 8 weeks (p<0.05). Although no biomechanical differences were observed, as expected for early healing, the results demonstrated that 2D RUST scoring significantly correlated with torsional biomechanics (p<0.01). In conclusion, 8-weeks of intermittent high-dose rPTH1-34 treatment significantly increases callus formation and accelerates bony union of intercalary massive allografts in a clinically relevant canine model, but with serious side-effects from hypercalcemia. PMID:29020057

Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing.

PubMed

Wang, Tao; Wang, Yongmei; Menendez, Alicia; Fong, Chak; Babey, Muriel; Tahimic, Candice G T; Cheng, Zhiqiang; Li, Alfred; Chang, Wenhan; Bikle, Daniel D

2015-09-01

Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1R(flox/flox) /2.3-kb α1(1)-collagen-Cre (KO) and IGF1R(flox/flox) (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by μCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation during fracture repair, but it plays an important role in coordinating chondrocyte, osteoclast, and endothelial responses that all contribute to the endochondral bone formation required for normal fracture repair. © 2015 American Society for Bone and Mineral Research.

Osteoblast-Specific Loss of IGF1R Signaling Results in Impaired Endochondral Bone Formation During Fracture Healing

PubMed Central

Wang, Tao; Wang, Yongmei; Menendez, Alicia; Fong, Chak; Babey, Muriel; Tahimic, Candice GT; Cheng, Zhiqiang; Li, Alfred; Chang, Wenhan; Bikle, Daniel D.

2017-01-01

Insulin-like growth factors (IGFs) are important local regulators during fracture healing. Although IGF1 deficiency is known to increase the risk of delayed union or non-union fractures in the elderly population, the underlying mechanisms that contribute to this defect remains unclear. In this study, IGF1 signaling during fracture healing was investigated in an osteoblast-specific IGF1 receptor (IGF1R) conditional knockout (KO) mouse model. A closed tibial fracture was induced in IGF1Rflox/flox/2.3-kb α1(1)-collagen-Cre (KO) and IGF1Rflox/flox (control) mice aged 12 weeks. Fracture callus samples and nonfractured tibial diaphysis were collected and analyzed by μCT, histology, immunohistochemistry, histomorphometry, and gene expression analysis at 10, 15, 21, and 28 days after fracture. A smaller size callus, lower bone volume accompanied by a defect in mineralization, bone microarchitectural abnormalities, and a higher cartilage volume were observed in the callus of these KO mice. The levels of osteoblast differentiation markers (osteocalcin, alkaline phosphatase, collagen 1α1) were significantly reduced, but the early osteoblast transcription factor runx2, as well as chondrocyte differentiation markers (collagen 2α1 and collagen 10α1) were significantly increased in the KO callus. Moreover, increased numbers of osteoclasts and impaired angiogenesis were observed during the first 15 days of fracture repair, but decreased numbers of osteoclasts were found in the later stages of fracture repair in the KO mice. Although baseline nonfractured tibias of KO mice had decreased trabecular and cortical bone compared to control mice, subsequent studies with mice expressing the 2.3-kb α1(1)-collagen-Cre ERT2 construct and given tamoxifen at the time of fracture and so starting with comparable bone levels showed similar impairment in fracture repair at least initially. Our data indicate that not only is the IGF1R in osteoblasts involved in osteoblast differentiation during fracture repair, but it plays an important role in coordinating chondrocyte, osteoclast, and endothelial responses that all contribute to the endochondral bone formation required for normal fracture repair. PMID:25801198

Optimization of methyl jasmonate and β-cyclodextrin for enhanced production of taraxerol and taraxasterol in (Taraxacum officinale Weber) cultures.

PubMed

Sharma, Kiran; Zafar, Rasheeduz

2016-06-01

Taraxacum officinale Weber (TO) commonly known as "dandelion", is a tropical Asian medicinal plant which contains taraxasterol (TX) and taraxerol (TA) in its roots, which are reported to be commercially important anticancer compounds. The main objective of the present study was to evaluate the increase in yield of TX and TA through elicitation by addition of abiotic elictors like methyl jasmonate (MJ) and β-cyclodextrin (CD), to the root callus suspension cultures of TO. The root callus suspension was maintained on Murashige and Skoog's (MS) medium MS + IAA + BA + 2, 4-D (0.5 ppm + 1 ppm + 0.5 ppm). The concentrations of the abiotic elicitors MJ and CD were optimized using central composite design (CCD) and quantification of TA and TX in elicited cultures was done by High Performance Liquid Chromatography (HPLC) analysis. It was observed that MJ at a concentration of 0.2 mM showed good increase in content of TX to 0.032% w/w and at concentrations 0.05 mM, 0.1 mM and 0.2 mM showed similar increase in TA content to 0.018% w/w, whereas CD at the concentration of 25 mM showed highest increase in TX content to 0.036% w/w and at the concentrations of 25 mM, 50 mM showed increase in TA content to 0.023% w/w as compared to the plant root (PR) which showed content of TX as 0.0299% w/w and TA as 0.0169% w/w. From the present investigation it was concluded that out of the two abiotic elicitors MJ and CD, CD was found to be more effective to increase TA and TX content in Dandelion cell cultures. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Preliminary results of in vitro propagation of Guayule

NASA Technical Reports Server (NTRS)

Dastoor, M. N.; Schubert, W. W.; Petersen, G. R.

1981-01-01

Guayule, Parthenium argentatum Gray, was first established in tissue culture by Bonner and Arreguin (1950) as a means to study the effects of various chemicals and extracts on rubber production. The propagation of whole guayule plants from tissue culture, however, has not been accomplished. The reported investigation is concerned with such an objective. In an attempt to stimulate rubber production in Guayule, Yokoyama et al. (1977) sprayed juvenile plants with 2-(3,4-dichloro-phenoxy)triethylamine (TEA derivative). This treatment resulted in increased isoprenoid levels in the plant tissue. In the current investigation, experiments were conducted to study the effect of TEA derivative on in vitro cultures. It was found that a suppression of callus formation occurs at a 10 mg/L concentration of TEA derivative with a resultant increase of shoot formation. Lower and higher concentrations of TEA derivative promote callus formation.

Phytosynthesis and Characterization of Silver Nanoparticles Using Callus of JATROPHA CURCAS: a Biotechnological Approach

NASA Astrophysics Data System (ADS)

Demissie, A. G.; Lele, S. S.

2013-06-01

The present study reports a rapid plant-based biosynthesis of silver nanoparticles using callus extract of Jatropha curcas L. The particle size and morphological analyses were carried out using Zetasizer, SEM, TEM. The physicochemical properties were monitored using UV-Vis spectroscopic, IR and DSC. The formation of silver nanoparticle was confirmed by using UV-Vis spectrophotometer and absorbance peaks at 421 nm. The silver nanoparticle was found to be a negatively charged with size ranging from 2 nm to 50 nm. The morphology of the nanoparticle is uniformly spherical and has a dispersion ratio of 0.14. The physicochemical study using DSC indicated significant thermal stability and crystalline nature of the nanoparticle. This intracellular biosynthesis of silver nanoparticles is simple, cheap and eco-friendly than other mechanical and chemical approaches.

Mandibular distraction osteogenesis with newly designed electromechanical distractor.

PubMed

Aykan, Andac; Ugurlutan, Rifat; Zor, Fatih; Ozturk, Serdar

2014-07-01

The purposes of this study were to design a fully automatic electromechanical distractor for continuous mandibular distraction osteogenesis and to investigate the efficacy of this newly developed distractor on sheep mandible model. Five sheep underwent unilateral mandibular osteotomy, and the mechanical component of electromechanical distractor was fixed on both sides of the osteotomy site using pins. After a 5-day latency period, the electromechanical distractor was activated at a rate of 0.30 mm per 8 hours using an electronic control unit. The bone was lengthened for 20 days without any intervention to the electromechanical distractor. The animals were killed on the sixth week of the consolidation period, and 5 distracted mandibles were examined through macroscopic observation and computed tomography. Distracted bone length was measured through computed tomography on sagittal slices. The device was tolerated by the distraction process without complications in all animals. New callus formation was observed on the distraction gap. Radiologic evaluation showed new callus formation in the distraction gap. New callus length was found to be, in average, 18.28 mm. In this preliminary study, a newly designed electromechanical distractor was successfully used for mandible distraction, which mainly provided a continuous lengthening during activation period spontaneously without any intervention. We think that the clinical application of this electromechanic distractor may provide patient comfort during distraction. Moreover, electromechanical distractor has the potential for high-resolution movement capacity when compared with annual distraction. The promising results from this prototype are encouraging to further investigations for human applications.

Sports-related dermatoses among road runners in Southern Brazil*

PubMed Central

Purim, Kátia Sheylla Malta; Leite, Neiva

2014-01-01

BACKGROUND Road running is a growing sport. OBJECTIVES: To determine the prevalence of sports-related dermatoses among road runners. METHODS Cross-sectional study of 76 road runners. Assessment was performed by means of a questionnaire, interview, and clinical examination. The chi-square and linear trend tests were used for analysis. RESULTS Most athletes were men (61%), aged 38±11 years, who ran mid- or long-distance courses (60.5%) for 45 to 60 minutes (79%), for a total of 25-64 km (42.1% ) or more than 65 km (18.4%) per week. The most prevalent injuries were blisters (50%), chafing (42.1%), calluses (34.2%), onychomadesis (31.5%), tinea pedis (18.4%), onychocryptosis (14.5%), and cheilitis simplex (14.5%). Among athletes running >64 km weekly, several conditions were significantly more frequent: calluses (p<0.04), jogger's nipple (p<0.004), cheilitis simplex (p<0.05), and tinea pedis (p<0.004). There was a significant association between the weekly running distance and the probability of skin lesions. Of the athletes in our sample, 57% trained before 10 a.m., 86% wore clothing and accessories for sun protection, 62% wore sunscreen, and 19.7% experienced sunburn. Traumatic and environmental dermatoses are common in practitioners of this outdoor sport, and are influenced by the weekly running distance. CONCLUSION In this group of athletes, rashes, blisters, sunburn, and nail disorders were recurrent complaints regardless of running distance. Calluses, athlete's foot, chapped lips, and jogger's nipple predominated in individuals who ran longer routes. PMID:25054745

Statistically optimized biotransformation protocol for continuous production of L-DOPA using Mucuna monosperma callus culture.

PubMed

Inamdar, Shrirang Appasaheb; Surwase, Shripad Nagnath; Jadhav, Shekhar Bhagwan; Bapat, Vishwas Anant; Jadhav, Jyoti Prafull

2013-01-01

L-DOPA (3,4-dihydroxyphenyl-L-alanine), a modified amino acid, is an expansively used drug for the Parkinson's disease treatment. In the present study, optimization of nutritional parameters influencing L-DOPA production was attempted using the response surface methodology (RSM) from Mucuna monosperma callus. Optimization of the four factors was carried out using the Box-Behnken design. The optimized levels of factors predicted by the model include tyrosine 0.894 g l(-1), pH 4.99, ascorbic acid 31.62 mg l(-1)and copper sulphate 23.92 mg l(-1), which resulted in highest L-DOPA yield of 0.309 g l(-1). The optimization of medium using RSM resulted in a 3.45-fold increase in the yield of L-DOPA. The ANOVA analysis showed a significant R (2) value (0.9912), model F-value (112.465) and probability (0.0001), with insignificant lack of fit. Optimized medium was used in the laboratory scale column reactor for continuous production of L-DOPA. Uninterrupted flow column exhibited maximum L-DOPA production rate of 200 mg L(-1) h(-1) which is one of the highest values ever reported using plant as a biotransformation source. L-DOPA production was confirmed by HPTLC and HPLC analysis. This study demonstrates the synthesis of L- DOPA using Mucuna monosperma callus using a laboratory scale column reactor.

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars.

PubMed

Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

2011-10-01

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1(-l)). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1(-1)) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N(6)-benzyladenine (BAP, 0.75 mg 1(-l)) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1(-l)) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.

High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.

PubMed

Lata, Hemant; Chandra, Suman; Khan, Ikhlas A; Elsohly, Mahmoud A

2010-10-01

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom. © Georg Thieme Verlag KG Stuttgart · New York.

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

PubMed Central

Aslam, Junaid; Khan, Saeed Ahmad; Cheruth, Abdul Jaleel; Mujib, Abdul; Sharma, Maheshwar Pershad; Srivastava, Prem Shanker

2011-01-01

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1−1) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N6-benzyladenine (BAP, 0.75 mg 1−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further. PMID:23961149

Proteomic identification of an embryo-specific 1Cys-Prx promoter and analysis of its activity in transgenic rice.

PubMed

Kim, Je Hein; Jung, In Jung; Kim, Dool Yi; Fanata, Wahyu Indra; Son, Bo Hwa; Yoo, Jae Yong; Harmoko, Rikno; Ko, Ki Seong; Moon, Jeong Chan; Jang, Ho Hee; Kim, Woe Yeon; Kim, Jae-Yean; Lim, Chae Oh; Lee, Sang Yeol; Lee, Kyun Oh

2011-04-29

Proteomic analysis of a rice callus led to the identification of 10 abscisic acid (ABA)-induced proteins as putative products of the embryo-specific promoter candidates. 5'-flanking sequence of 1 Cys-Prx, a highly-induced protein gene, was cloned and analyzed. The transcription initiation site of 1 Cys-Prx maps 96 nucleotides upstream of the translation initiation codon and a TATA-box and putative seed-specific cis-acting elements, RYE and ABRE, are located 26, 115 and 124 bp upstream of the transcription site, respectively. β-glucuronidase (GUS) expression driven by the 1 Cys-Prx promoters was strong in the embryo and aleurone layer and the activity reached up to 24.9 ± 3.3 and 40.5 ± 2.1 pmol (4 MU/min/μg protein) in transgenic rice seeds and calluses, respectively. The activity of the 1 Cys-Prx promoters is much higher than that of the previously-identified embryo-specific promoters, and comparable to that of strong endosperm-specific promoters in rice. GUS expression driven by the 1 Cys-Prx promoters has been increased by ABA treatment and rapidly induced by wounding in callus and at the leaf of the transgenic plants, respectively. Furthermore, ectopic expression of the GUS construct in Arabidopsis suggested that the 1 Cys-Prx promoter also has strong activity in seeds of dicot plants. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene expression patterns during somatic embryo development and germination in maize Hi II callus cultures.

PubMed

Che, Ping; Love, Tanzy M; Frame, Bronwyn R; Wang, Kan; Carriquiry, Alicia L; Howell, Stephen H

2006-09-01

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an alpha-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.

Objective guidelines for removing an external fixator after tibial lengthening using pixel value ratio: a pilot study.

PubMed

Zhao, Li; Fan, Qing; Venkatesh, K P; Park, Man S; Song, Hae Ryong

2009-12-01

During limb lengthening over an intramedullary nail, decisions regarding external fixator removal and weightbearing depend on the amount of callus seen at the lengthening area on radiographs. However, this method is subjective and objective evaluation of the amount of callus likely would minimize nail or interlocking screw breakage and refracture after fixator removal. We asked how many cortices with full corticalization of the newly formed bone at the lengthening area are needed to allow fixator removal and full weightbearing and how to radiographically determine the stage of corticalization. We retrospectively reviewed 17 patients (34 lengthenings) who underwent bilateral tibial lengthenings over an intramedullary nail. The average gain in length was 7.2 +/- 3.4 cm. We determined the pixel value ratio (ratio of pixel value of regenerate versus the mean pixel value of adjacent bone) of the lengthened area on radiographs. There were no nail or screw breakage and refracture. Partial weightbearing with crutches was permitted when the pixel value ratio was 1 in two cortices and full weightbearing without crutches was permitted when the pixel value ratio was 1 in three cortices. The pixel value ratio on radiographs can be an objective parameter for callus measurement and may provide guidelines for the timing of external fixator removal. We cannot determine from our limited data the minimum pixel value in how many cortices would suggest safe removal, but we can say our criteria were not associated with subsequent refracture.

Effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs.

PubMed

Ochi, Hiroki; Hara, Yasushi; Asou, Yoshinori; Harada, Yasuji; Nezu, Yoshinori; Yogo, Takuya; Shinomiya, Kenichi; Tagawa, Masahiro

2011-05-01

To evaluate effects of long-term administration of carprofen on healing of a tibial osteotomy in dogs. 12 healthy female Beagles. A mid-diaphyseal transverse osteotomy (stabilized with an intramedullary pin) of the right tibia was performed in each dog. The carprofen group (n = 6 dogs) received carprofen (2.2 mg/kg, PO, q 12 h) for 120 days; the control group (6) received no treatment. Bone healing and change in callus area were assessed radiographically over time. Dogs were euthanized 120 days after surgery, and tibiae were evaluated biomechanically and histologically. The osteotomy line was not evident in the control group on radiographs obtained 120 days after surgery. In contrast, the osteotomy line was still evident in the carprofen group. Callus area was significantly less in the carprofen group, compared with the area in the control group, at 20, 30, and 60 days after surgery. At 120 days after surgery, stiffness, elastic modulus, and flexural rigidity in the carprofen group were significantly lower than corresponding values in the control group. Furthermore, histologic evaluation revealed that the cartilage area within the callus in the carprofen group was significantly greater than that in the control group. Long-term administration of carprofen appeared to inhibit bone healing in dogs that underwent tibial osteotomy. We recommend caution for carprofen administration when treating fractures that have delays in healing associated with a reduction in osteogenesis as well as fractures associated with diseases that predispose animals to delays of osseous repair.

Arabidopsis WPP-Domain Proteins Are Developmentally Associated with the Nuclear Envelope and Promote Cell DivisionW⃞

PubMed Central

Patel, Shalaka; Rose, Annkatrin; Meulia, Tea; Dixit, Ram; Cyr, Richard J.; Meier, Iris

2004-01-01

The nuclear envelope (NE) acts as a selective barrier to macromolecule trafficking between the nucleus and the cytoplasm and undergoes a complex reorganization during mitosis. Different eukaryotic kingdoms show specializations in NE function and composition. In contrast with vertebrates, the protein composition of the NE and the function of NE proteins are barely understood in plants. MFP1 attachment factor 1 (MAF1) is a plant-specific NE-associated protein first identified in tomato (Lycopersicon esculentum). Here, we demonstrate that two Arabidopsis thaliana MAF1 homologs, WPP1 and WPP2, are associated with the NE specifically in undifferentiated cells of the root tip. Reentry into cell cycle after callus induction from differentiated root segments reprograms their NE association. Based on green fluorescent protein fusions and immunogold labeling data, the proteins are associated with the outer NE and the nuclear pores in interphase cells and with the immature cell plate during cytokinesis. RNA interference–based suppression of the Arabidopsis WPP family causes shorter primary roots, a reduced number of lateral roots, and reduced mitotic activity of the root meristem. Together, these data demonstrate the existence of regulated NE targeting in plants and identify a class of plant-specific NE proteins involved in mitotic activity. PMID:15548735

How to Treat Corns and Calluses

MedlinePlus

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Novel potato plants with enhanced cadmium resistance and antioxidative defence generated after in vitro cell line selection.

PubMed

Ashrafzadeh, Seyedardalan; Leung, David W M

2017-01-01

It is of interest to apply plant tissue culture to generate plants resistant to toxic effects of cadmium (Cd) on plant growth. Callus cultures were initiated from leaf explants of micropropagated potato plantlets (Solanum tuberosum L., cv. Iwa) for in vitro selection comprising 18 different Cd treatments varying in Cd exposure timing and duration. Plantlets regenerated from two different lines of Cd-selected calli, L9 and L11, were found to exhibit enhanced resistance to 218 μM Cd compared to control (source plantlets for leaf explants used to initiate callus cultures for Cd resistance). In response to 218 μM Cd, L11 plantlets had lower levels of lipid peroxidation and hydrogen peroxide than control and L9 plantlets. In addition, antioxidative enzyme activities in L11 were generally higher than control. L11 also had a higher level of proline than control.

Variation in the accumulation levels of N,N-dimethyltryptamine in micropropagated trees and in in vitro cultures of Mimosa tenuiflora.

PubMed

Nicasio, María Del Pilar; Villarreal, María Luisa; Gillet, Françoise; Bensaddek, Lamine; Fliniaux, Marc-André

2005-01-01

The present article reports the accumulation of N,N-dimethyltryptamine and its metabolic precursors (tryptophan, tryptamine) in different organs of micropropagated Mimosa tenuiflora trees (leaves, flowers and bark) subjected to seasonal variations (January and June), as well as in in vitro cultures (plantlets and calluses) of this plant species. The accumulation of all the tested compounds varied according to the organ, the month of collection, and age of the plant material. In all cases, the neurotoxic compound N,N-dimethyltryptamine (DMT) was detected with the lowest concentration 0.01% dry weight (DW) in flowers, and the highest 0.33% DW in bark. For the in vitro cultures, DMT was present in high yields in plantlets (0.1-0.2% DW), while in calluses this compound was initially detected but its concentration decreased significantly in the subsequent subcultures.

Water-Wisteria as an ideal plant to study heterophylly in higher aquatic plants.

PubMed

Li, Gaojie; Hu, Shiqi; Yang, Jingjing; Schultz, Elizabeth A; Clarke, Kurtis; Hou, Hongwei

2017-08-01

The semi-aquatic plant Water-Wisteria is suggested as a new model to study heterophylly due to its many advantages and typical leaf phenotypic plasticity in response to environmental factors and phytohormones. Water-Wisteria, Hygrophila difformis (Acanthaceae), is a fast growing semi-aquatic plant that exhibits a variety of leaf shapes, from simple leaves to highly branched compound leaves, depending on the environment. The phenomenon by which leaves change their morphology in response to environmental conditions is called heterophylly. In order to investigate the characteristics of heterophylly, we assessed the morphology and anatomy of Hygrophila difformis in different conditions. Subsequently, we verified that phytohormones and environmental factors can induce heterophylly and found that Hygrophila difformis is easily propagated vegetatively through either leaf cuttings or callus induction, and the callus can be easily transformed by Agrobacterium tumefaciens. These results suggested that Hygrophila difformis is a good model plant to study heterophylly in higher aquatic plants.

Black bone disease in a healing fracture.

PubMed

Thiam, Desmond; Teo, Tse Yean; Malhotra, Rishi; Tan, Kong Bing; Chee, Yu Han

2016-01-28

Black bone disease refers to the hyperpigmentation of bone secondary to prolonged usage of minocycline. We present a report of a 34-year-old man who underwent femoral shaft fracture fixation complicated by deep infection requiring debridement. The implants were removed 10 months later after long-term treatment with minocycline and fracture union. A refracture of the femoral shaft occurred 2 days after implant removal and repeat fixation was required. Intraoperatively, abundant heavily pigmented and dark brown bone callus was noted over the old fracture site. There was no evidence of other bony pathology and the appearance was consistent with minocycline-associated pigmentation. As far as we are aware, this is the first case of black bone disease affecting callus within the interval period of bone healing. We also discuss the relevant literature on black bone disease to bring light on this rare entity that is an unwelcomed surprise to operating orthopaedic surgeons. 2016 BMJ Publishing Group Ltd.

Effect of selection agents to Chrysanthemum (Chrysanthemum morifolium) callus growth after Agrobacterium-mediated genetic transformation

NASA Astrophysics Data System (ADS)

Sjahril, R.; Jamaluddin, I.; Nadir, M.; Asman; Dungga, N. E.

2018-05-01

Genetic transformation mediated by Agrobacterium tumefaciens requires an efficient selection method for successful progress of transformation. This study aims to determine the concentration and kind of antibiotics and selection agents used during transformation to formulate standard protocol of chrysanthemum in the process of propagating disease resistant Chrysanthemum mediated by Agrobacterium tumefaciens EHA105 (pEKB-WD). The experiments were performed by planting chrysanthemum explants leaf cutting (5 mm diameter on NAA medium 2 mg L-1 BAP 2 mg L-1) with addition of Kanamycin: 25, 50, 100, 150 and 200 (mg L-1); Hygromycin: 5, 10, 25, 50 and 75 (mg L-1); Paromomycin: 10, 25, 50, 75 and 100 (mg L-1). Experiment was arranged in a Completely Randomized Design (CRD). Each treatment was repeated five times thus 75 bottles of culture were used; each bottle consists of 5 pieces of leaf cuttings, resulted in total of 375 pieces. The results showed that selection agent had a critical value for Hygromycin 25 mg L-1 and Kanamycin 100 mg L-1 which can make explant experienced necrosis better than Paromomycin. Paromomycin at 100 mg L-1 was only able to kill explant’s periphery. Remained callus stayed fresh more than 50% so that when used as the selection agent could produce more escape cell. The optimum transformation with concentration of 10% Agrobacterium (vol/vol) with 30 minutes co-cultivation can produce more efficient transformed callus. Considering the high price of Hygromycin, it was best to use Kanamycin as selective agents.

Induction of a photomixotrophic plant cell culture of Helianthus annuus and optimization of culture conditions for improved α-tocopherol production.

PubMed

Geipel, Katja; Song, Xue; Socher, Maria Lisa; Kümmritz, Sibylle; Püschel, Joachim; Bley, Thomas; Ludwig-Müller, Jutta; Steingroewer, Juliane

2014-03-01

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used, e.g., as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l(-1) sucrose, 0.5 mg l(-1) of the auxin 1-naphthalene acetic acid, and 0.5 mg l(-1) of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration, and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230%) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.

Intermittent parathyroid hormone administration improves periodontal healing in rats.

PubMed

Vasconcelos, Daniel Fernando Pereira; Marques, Marcelo Rocha; Benatti, Bruno Braga; Barros, Silvana Pereira; Nociti, Francisco Humberto; Novaes, Pedro Duarte

2014-05-01

Intermittent administration of parathyroid hormone (PTH) promotes new bone formation in patients with osteoporosis and bone fractures. It was shown previously that PTH also reduces periodontitis-related bone loss. The aim of this study is to evaluate the effect of treatment with PTH on periodontal healing in rats. Fenestration defects were created at the buccal surface of the distal root of the mandibular first molars, and both periodontal ligament (PDL) and cementum were removed. Animals were then assigned to two groups (eight animals per group): group 1: control, placebo administration; and group 2: test, human PTH (hPTH) 1-34 administration at a concentration of 40 μg/kg. For both groups, the animals were injected every 2 days, and the animals were sacrificed at 14 and 21 days after surgery. Specimens were harvested and processed for routine decalcified histologic sections. The following parameters were assessed: 1) remaining bone defect extension (RBDE); 2) newly formed bone density (NFBD); 3) total callus area (TCA); 4) osteoclast number (ON) in the callus region; and 5) newly formed dental cementum-like tissue (NFC). Birefringence of root PDL reattachment was also evaluated. Birefringence analysis showed root PDL reattachment for both groups 21 days after treatment. Intermittent hPTH 1-34 administration decreased RBDE (P <0.01) and increased NFBD (P <0.01), TCA (P <0.01), area of NFC (P <0.01), and ON in the callus region (P <0.01). Within the limits of the present study, intermittent administration of hPTH 1-34 led to an enhanced periodontal healing process compared with non-treated animals.

Beneficial effects of foot care nursing for people with diabetes mellitus: an uncontrolled before and after intervention study.

PubMed

Fujiwara, Yuko; Kishida, Ken; Terao, Mika; Takahara, Mitsuyoshi; Matsuhisa, Munehide; Funahashi, Tohru; Shimomura, Iichiro; Shimizu, Yasuko

2011-09-01

The aim of this study was to assess the effectiveness of a preventative foot care nursing programme for diabetic patients. Foot complications are common in diabetic patients and prevention of such complications requires foot care. However, there is little information on the effectiveness of foot care nursing on the incidence and recurrence of diabetic foot. We developed a diabetic foot care programme based on the International Working Group on the Diabetic Foot. We studied 88 patients who attended our foot care programme for 2 years, and collected data from April 2005 to March 2009. Patients were divided into four groups according to the risk classification, and received foot care. We evaluated the incidence of foot ulceration or recurrence and non-ulcerated foot condition. Characteristics of the patients were analysed using the paired t-test and McNemar's test, and changes in severity of tinea pedis and grade of callus were analysed using Wilcoxon's signed rank sum test. The programme reduced the severity score of tinea pedis (P < 0·001) and improved callus grade (P < 0·001). All these were evaluated by Wilcoxon's signed rank sum test. None of the patients of risk-group-3 (history of foot ulceration) showed recurrence of callus-related foot ulcers. Six high-risk patients developed foot ulceration during the programme because of minor injury, but the ulcers healed without development of gangrene. A nurse-based foot care programme is effective in preventing diabetic foot in diabetic patients. © 2011 The Authors. Journal of Advanced Nursing © 2011 Blackwell Publishing Ltd.

New alternative in treatment of callus.

PubMed

Akdemir, Ovunc; Bilkay, Ufuk; Tiftikcioglu, Yigit Ozer; Ozek, Cüneyt; Yan, Hede; Zhang, Feng; Akin, Yalcin

2011-02-01

The pathological transformation of the skin into a thick and hard callus due to repetitive trauma or friction is commonly known as corn. Although a variety of medical and operative treatment choices have been proposed, an ideal treatment method is yet to be defined. Effectiveness of tangential excision together with topical cantharidin has been evaluated. We used Canthacur-PS as an adjunct to excision in an outpatient setting. Canthacur-PS is a commercially available topical solution that includes 1% cantharidin, 30% salicylic acid and 5% podophyllin. The treatment has been applied to 72 patients. We found that 65 patients (90.3%) had corn on their feet and seven patients (9.7%) on their hands. Thick, hard and hyperkeratotic skin area was scraped with the help of a no. 15 blade. The solution was applied on and around the periphery (up to 1–2 mm) of the lesion with a cotton swab, and kept closed for 5 days with an antibiotic dressing. All the patients had been followed up for at least 1 year and evaluated by clinical examination and patient satisfaction query. One session of treatment succeeded in 57 (79.2%) corn patients. Two sessions in nine corn patients (12.5%), three sessions in five corn patients (6.9%) and four sessions in one patient (1.4%) were needed. Only one recurrence (1.4%) was seen. No scar formation or other side-effects were seen. Our findings show that this treatment method is a simple, minimally invasive and reliable treatment for calluses. © 2010 Japanese Dermatological Association.

Cannabidiol, a Major Non-Psychotropic Cannabis Constituent Enhances Fracture Healing and Stimulates Lysyl Hydroxylase Activity in Osteoblasts.

PubMed

Kogan, Natalya M; Melamed, Eitan; Wasserman, Elad; Raphael, Bitya; Breuer, Aviva; Stok, Kathryn S; Sondergaard, Rachel; Escudero, Ana V Villarreal; Baraghithy, Saja; Attar-Namdar, Malka; Friedlander-Barenboim, Silvina; Mathavan, Neashan; Isaksson, Hanna; Mechoulam, Raphael; Müller, Ralph; Bajayo, Alon; Gabet, Yankel; Bab, Itai

2015-10-01

Cannabinoid ligands regulate bone mass, but skeletal effects of cannabis (marijuana and hashish) have not been reported. Bone fractures are highly prevalent, involving prolonged immobilization and discomfort. Here we report that the major non-psychoactive cannabis constituent, cannabidiol (CBD), enhances the biomechanical properties of healing rat mid-femoral fractures. The maximal load and work-to-failure, but not the stiffness, of femurs from rats given a mixture of CBD and Δ(9) -tetrahydrocannabinol (THC) for 8 weeks were markedly increased by CBD. This effect is not shared by THC (the psychoactive component of cannabis), but THC potentiates the CBD stimulated work-to-failure at 6 weeks postfracture followed by attenuation of the CBD effect at 8 weeks. Using micro-computed tomography (μCT), the fracture callus size was transiently reduced by either CBD or THC 4 weeks after fracture but reached control level after 6 and 8 weeks. The callus material density was unaffected by CBD and/or THC. By contrast, CBD stimulated mRNA expression of Plod1 in primary osteoblast cultures, encoding an enzyme that catalyzes lysine hydroxylation, which is in turn involved in collagen crosslinking and stabilization. Using Fourier transform infrared (FTIR) spectroscopy we confirmed the increase in collagen crosslink ratio by CBD, which is likely to contribute to the improved biomechanical properties of the fracture callus. Taken together, these data show that CBD leads to improvement in fracture healing and demonstrate the critical mechanical role of collagen crosslinking enzymes. © 2015 American Society for Bone and Mineral Research.

Application of a novel bone osteotomy plate leads to reduction in heat-induced bone tissue necrosis in sheep.

PubMed

Bekić, Marijo; Davila, Slavko; Hrskanović, Mato; Bekić, Marijana; Seiwerth, Sven; Erdeljić, Viktorija; Capak, Darko; Butković, Vladimir

2008-12-01

Previous studies have shown substantial effect thermal damage can have on new bone formation following osteotomy. In this study we evaluated the extent of thermal damage which occurs in four different methods of osteotomy and the effects it can have on bone healing. We further wanted to test whether a special osteotomy plate we constructed can lead to diminished heat generation during osteotomy and enhanced bone healing. The four methods evaluated included osteotomy performed by chisel, a newly constructed osteotomy plate, Gigly and oscillating saw. Twelve adult sheep underwent osteotomy performed on both tibiae. Bone fragments were stabilized using a fixation plate. Callus size was assessed using standard radiographs. Densitometry and histological evaluation were performed at 8 weeks following osteotomy. Temperature measurements were performed both in vivo during the operation, and ex vivo on explanted tibiae. The defects healed without complications and showed typical course of secondary fracture healing with callus ingrowth into the osteotomy gap. Radiographic examination of bone healing showed a tendency towards more callus formation in bones osteotomized using Gigly and oscillating saw, but this difference lacked significance. Use of Gigly and oscillating saw elicited much higher temperatures at the bone cortex surface, which subsequently lead to slightly impaired bone healing according to histological analysis. BMD was equal among all bones. In conclusion, the time required for complete healing of the defect differed depended greatly on the instruments used. The newly constructed osteotomy plate showed best results based on histological findings of capillary and osteoblast density.

Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro

PubMed Central

Ma, Jun; He, Yehua; Hu, Zhongyi; Xu, Wentian; Xia, Jingxian; Guo, Cuihong; Lin, Shunquan; Chen, Chengjie; Wu, Chenghou; Zhang, Junli

2014-01-01

Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20–80 fold more than in anther but similar to that of non-embryogenic callus, which was 20–50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity. PMID:25249776

The Effects of Herbicides Targeting Aromatic and Branched Chain Amino Acid Biosynthesis Support the Presence of Functional Pathways in Broomrape.

PubMed

Dor, Evgenia; Galili, Shmuel; Smirnov, Evgeny; Hacham, Yael; Amir, Rachel; Hershenhorn, Joseph

2017-01-01

It is not clear why herbicides targeting aromatic and branched-chain amino acid biosynthesis successfully control broomrapes-obligate parasitic plants that obtain all of their nutritional requirements, including amino acids, from the host. Our objective was to reveal the mode of action of imazapic and glyphosate in controlling the broomrape Phelipanche aegyptiaca and clarify if this obligatory parasite has its own machinery for the amino acids biosynthesis. P. aegyptiaca callus was studied to exclude the indirect influence of the herbicides on the parasite through the host plant. Using HRT - tomato plants resistant to imidazolinone herbicides, it was shown that imazapic is translocated from the foliage of treated plants to broomrape attachments on its roots and controls the parasite. Both herbicides inhibited P. aegyptiaca callus growth and altered the free amino acid content. Blasting of Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS) cDNA against the genomic DNA of P. aegyptiaca yielded a single copy of each homolog in the latter, with about 78 and 75% similarity, respectively, to A. thaliana counterparts at the protein level. We also show for the first time that both EPSPS and ALS are active in P. aegyptiaca callus and flowering shoots and are inhibited by glyphosate and imazapic, respectively. Thus leading to deficiency of those amino acids in the parasite tissues and ultimately, death of the parasite, indicating the ability of P. aegyptiaca to synthesize branched-chain and aromatic amino acids through the activity of ALS and EPSPS, respectively.

The Effects of Herbicides Targeting Aromatic and Branched Chain Amino Acid Biosynthesis Support the Presence of Functional Pathways in Broomrape

PubMed Central

Dor, Evgenia; Galili, Shmuel; Smirnov, Evgeny; Hacham, Yael; Amir, Rachel; Hershenhorn, Joseph

2017-01-01

It is not clear why herbicides targeting aromatic and branched-chain amino acid biosynthesis successfully control broomrapes—obligate parasitic plants that obtain all of their nutritional requirements, including amino acids, from the host. Our objective was to reveal the mode of action of imazapic and glyphosate in controlling the broomrape Phelipanche aegyptiaca and clarify if this obligatory parasite has its own machinery for the amino acids biosynthesis. P. aegyptiaca callus was studied to exclude the indirect influence of the herbicides on the parasite through the host plant. Using HRT – tomato plants resistant to imidazolinone herbicides, it was shown that imazapic is translocated from the foliage of treated plants to broomrape attachments on its roots and controls the parasite. Both herbicides inhibited P. aegyptiaca callus growth and altered the free amino acid content. Blasting of Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS) cDNA against the genomic DNA of P. aegyptiaca yielded a single copy of each homolog in the latter, with about 78 and 75% similarity, respectively, to A. thaliana counterparts at the protein level. We also show for the first time that both EPSPS and ALS are active in P. aegyptiaca callus and flowering shoots and are inhibited by glyphosate and imazapic, respectively. Thus leading to deficiency of those amino acids in the parasite tissues and ultimately, death of the parasite, indicating the ability of P. aegyptiaca to synthesize branched-chain and aromatic amino acids through the activity of ALS and EPSPS, respectively. PMID:28523011

DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

PubMed

Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

2017-06-01

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Testosterone Delivered with a Scaffold Is as Effective as Bone Morphologic Protein-2 in Promoting the Repair of Critical-Size Segmental Defect of Femoral Bone in Mice

PubMed Central

Cheng, Bi-Hua; Chu, Tien-Min G.; Chang, Chawnshang; Kang, Hong-Yo; Huang, Ko-En

2013-01-01

Loss of large bone segments due to fracture resulting from trauma or tumor removal is a common clinical problem. The goal of this study was to evaluate the use of scaffolds containing testosterone, bone morphogenetic protein-2 (BMP-2), or a combination of both for treatment of critical-size segmental bone defects in mice. A 2.5-mm wide osteotomy was created on the left femur of wildtype and androgen receptor knockout (ARKO) mice. Testosterone, BMP-2, or both were delivered locally using a scaffold that bridged the fracture. Results of X-ray imaging showed that in both wildtype and ARKO mice, BMP-2 treatment induced callus formation within 14 days after initiation of the treatment. Testosterone treatment also induced callus formation within 14 days in wildtype but not in ARKO mice. Micro-computed tomography and histological examinations revealed that testosterone treatment caused similar degrees of callus formation as BMP-2 treatment in wildtype mice, but had no such effect in ARKO mice, suggesting that the androgen receptor is required for testosterone to initiate fracture healing. These results demonstrate that testosterone is as effective as BMP-2 in promoting the healing of critical-size segmental defects and that combination therapy with testosterone and BMP-2 is superior to single therapy. Results of this study may provide a foundation to develop a cost effective and efficient therapeutic modality for treatment of bone fractures with segmental defects. PMID:23940550

Physiological Disorders of Pear Shoot Cultures

USDA-ARS?s Scientific Manuscript database

Physiological disorders are some of the most difficult challenges in micropropagation. Little is known of the causes of plant growth disorders which include callus formation, hyperhydricity, shoot tip necrosis, leaf lesions, epinasty, fasciation and hypertrophy. During our study of mineral nutritio...

Bunions

MedlinePlus

... toe turned toward the other toes and may cross over the second toe. Corns and calluses develop as a result where the first and second toes overlap. Difficulty wearing regular shoes. You may have problems finding shoes that fit or that do not cause pain.

Oligosaccharins.

ERIC Educational Resources Information Center

Albersheim, Peter; Darvill, Alan G.

1985-01-01

Related history and laboratory work which lead to isolation of oligosaccharins, a new class of regulatory molecules found in plant cell walls. These substances function in growth, development, reproduction, and defense. Mixtures of oligosaccharins and other hormones can stimulate growth of an undifferentiated callus, roots, vegetative…

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana).

PubMed

Jones, A Maxwell P; Chattopadhyay, Abhishek; Shukla, Mukund; Zoń, Jerzy; Saxena, Praveen K

2012-05-30

Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

PubMed Central

2012-01-01

Background Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division. Results This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. Conclusions This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts. PMID:22646730

Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

PubMed

Garrocho-Villegas, Verónica; Aguilar C, Raúl; Sánchez de Jiménez, Estela

2013-12-23

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Somatic Embryogenesis in Lisianthus (Eustoma russellianum Griseb.).

PubMed

Ruffoni, Barbara; Bassolino, Laura

2016-01-01

Somatic embryogenesis is, for the main floricultural crops, a promising system for commercial scale-up, providing cloned material to be traded as seedlings. Somatic embryos, having the contemporary presence of root apical meristem and shoot apical meristem, can be readily acclimatized. For Lisianthus it is possible to induce embryogenic callus from leaf fragments of selected genotypes and to obtain embryos either in agarized substrate or in liquid suspension culture. The production of somatic embryos in liquid medium is high and can be modulated in order to synchronize the cycle and the size of the neoformed structures. The possibility to use the liquid substrate with high propagation rates reduces labor costs and could support the costs of eventual automation. In this paper we report a stepwise protocol for somatic embryogenesis in the species Eustoma russellianum.

Footwear

MedlinePlus

... the Big Toe Ailments of the Smaller Toes Diabetic Foot Treatments Injections and other Procedures Treatments of the ... Shoe IQ How to "Read" Your Shoes Custom Diabetic Shoes 10 Points of Proper Shoe Fit ... Footwear Page Content Do you experience disabling foot problems like bunions, corns, calluses or hammer toes? ...

29 CFR 776.2 - Employee basis of coverage.

Code of Federal Regulations, 2010 CFR

2010-07-01

... in covered work, their employer cannot avoid his obligations to them under the Act on the ground that.... Callus, 325 U.S. 578; Armour & Co. v. Wantock, 323 U.S. 126; Donovan v. Shell Oil Co., 168 F. 2d 229 (C.A...

The effects of nail rigidity on fracture healing in rats with osteoporosis

PubMed Central

Sha, Mo; Fu, Jun; Li, Jing; Fan Yuan, Chao; Shi, Lei; Jun Li, Shu

2009-01-01

Background and purpose Stress shielding from rigid internal fixation may lead to refracture after removal of the osteosynthesis material. We investigated the effect of a low-rigidity (Ti-24Nb-4Zr-7.9Sn) intramedullary nail regarding stress shielding and bone healing of osteoporotic fractures in the rat. Methods 40 female Sprague-Dawley rats, aged 3 months, were divided into the following groups: sham-operation (SHAM) (n = 10), ovariectomized (OVX) (n = 10) and OVX-fracture (n = 20). 10 SHAM rats and 10 OVX rats were killed after 12 weeks to provide biomechanical data. Ovariectomy was performed 12 weeks before fracturing both femurs in 20 rats. The left fracture was stabilized with a high-rigidity titanium alloy pin (Ti-6Al-4V; elastic modulus 110 GPa) and the right with a low-rigidity (Ti-24Nb-4Zr-7.9Sn; elastic modulus 33 GPa). The bony calluses were examined by micro-CT at 6 and 12 weeks after fracture, bone volume (BV) and total volume (TV) were determined at the callus region (ROI1) and the total femur (ROI2). Subsequently, the bones were tested mechanically by a three-point bending test. Results In the low-rigidity group, TV (ROI1) increased at 6 weeks, but BV (ROI1), BV (ROI2) were similar but maximum load increased. At 12 weeks, the maximum load and also BV (ROI1, ROI2) were increased in the low-rigidity group. Interpretation The low-rigidity nail manufactured from Ti-24Nb-4Zr-7.9Sn showed better external callus formation, seemed to reduce effects of stress shielding, and reduced bone resorption better than the stiffer nail. The low-rigidity nail was strong enough to maintain alignment of the fracture in the osteoporotic rat model without delayed union. PMID:19297794

Morphologic and molecular alteration during tibia fracture healing in rat.

PubMed

Yu, M-D; Su, B-H; Zhang, X-X

2018-03-01

To monitor morphological feature and related osteogenic and bone metabolic change during healing of tibia fracture in a rat model. Tibia density and trabecular thickness were evaluated. Histopathology was examined by HE staining. Serous inflammatory factors IL-4, IL-6, TNF-α and metabolic biomarkers ALP, β-CTX, P1NP, were determined by ELISA. The expression of RUNX2, TGF-β1, VEGF-α, BMP-2, BMP-4, and BMP-7 in callus tissue were qualified by RT-PCR. Bone density decreased until week 4 and then increased post-operation. Trabeculae in callus were thickened over time with active osteogenesis. ELISA indicated the most severe inflammation at week 2, with the highest level of TNF-α, IL-6, and the lowest level of IL-4. After 4 weeks, the inflammation was alleviated accompanying with the decline of TNF-α and IL-6, while there was the elevation of IL-4. Bone metabolism showed active osteogenesis and resorption at week 6 with high P1NP and β-CTX. The expression of RUNX2, TGF-β1, VEGF-α, BMP-2, BMP-4, and BMP-7 increased progressively from week 1 to 6. The major lesions at week 2 in sham were tissue necrosis, periosteal reactive hyperplasia, inflammatory cell infiltration, capillary hyperplasia and slight fibro-blast cytopoiesis. At week 4, proliferation was greatly activated, fibrous callus shaped and chondrogenesis and some osteogenesis occurred at week 8. In rat model, bone density started to increase at week 6 after fracture, accompanied with trabeculae thickening, serous inflammatory factors decline, and peaked bone morphogenetic protein/growth factors, which indicated active osteogenesis was conforming to the classical phase of secondary fracture healing.

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

PubMed Central

Rikiishi, Kazuhide; Matsuura, Takakazu; Ikeda, Yoko; Maekawa, Masahiko

2015-01-01

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars. PMID:26670930

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

PubMed

Peña, Leandro; Pérez, Rosa M; Cervera, Magdalena; Juárez, José A; Navarro, Luis

2004-07-01

Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Establishment of an Arabidopsis callus system to study the interrelations of biosynthesis, degradation and accumulation of carotenoids

PubMed Central

Schaub, Patrick; Rodriguez-Franco, Marta; Cazzonelli, Christopher Ian; Álvarez, Daniel; Wüst, Florian

2018-01-01

The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops. PMID:29394270

A survey of injection site lesions in fed cattle in Canada.

PubMed Central

Van Donkersgoed, J; Dixon, S; Brand, G; VanderKop, M

1997-01-01

During November 1996 to January 1997, a survey was conducted at 5 Canadian purveyors to measure the prevalence of injection site lesions in the top butt, boneless blade, outside round, inside round, and eye of the round. As trimmers were cutting these subprimals into steaks, technicians monitored each steak for grossly obvious scars. These scars were trimmed, weighed, and scored as either a "clear scar," "woody callus," or "cyst." All scars were subsequently examined histologically and classified as a "clear scar," "woody callus," "scar with nodules," "mineralized scar," or "cyst." Pieces were observed for broken needles while being processed and none were found. The estimated prevalence of injection site lesions was 18.8% (95% CI, 16.4% to 21.2%) in top butts, 22.2% (95% CI, 18.8% to 25.7%) in boneless blades, 4.9% (95% CI, 3.6% to 6.3%) in the eye of round, 1.8% (95% CI, 1.1% to 2.9%) in the inside round, and 7.6% (95% CI, 5.6% to 9.8%) in the outside round. Some top butts originated from American fed cattle; the estimated prevalence of lesions was 9.0% (95% CI, 5.9% to 12.9%) in American top butts and 22.3% (95% CI, 19.4% to 25.3%) in Canadian top butts. The median weight of the lesions varied among subprimals and ranged from 64 g to 117 g. Histologically, 13% of the scars were clear scars, 47% were woody calluses, 5% were mineralized scars, 34% were scars with nodules, 0.2% were cysts, and 0.9% were normal fat infiltrations. An economic analysis estimated an average loss of $8.95 per fed animal processed or $19 million dollars annually to the Canadian beef industry from injection scars. PMID:9426942

Impaired joint motion and contractures in callus distraction and segment transport: a retrospective data analysis.

PubMed

Zak, Lukas; Wozasek, Gerald E

2013-11-01

The temporary loss of motion of adjacent joints is a common complication after distraction osteogenesis of the lower limb. The aim of this study was to investigate the incidence of tendon contracture and impaired joint motion of the knee and/or ankle joint during and after callus distraction with a ring fixator. Twenty patients (2 female, 18 male, average age: 36 years) were surgically treated for callus distraction and segment transport with an external ring fixator after traumatic bone loss in 21 lower limbs. The impaired joint motion of the adjacent joints during and after treatment was evaluated. During treatment, we observed the free range of motion (ROM) of the ankle joint in 4 cases (19 %), restricted motion in 11 cases (52 %), and complete loss of motion in 6 cases (33 %). After treatment,free ROM was observed in 12 cases (57 %), impaired motion in 3 cases (14 %), and fixed joint position in 6 cases (29 %, 2 arthrodesis). This represents an improvement of motion in eight cases (38 %) and an impairment in two cases (10 %). In 11 cases, the ROM remained unchanged. During treatment, six restrictions in extension (24 %) and five (33 %) restrictions in flexion occurred in the knee joint, ultimately resulting in one loss of extension and three losses of flexion after frame removal. The impairment of joint motion during bone lengthening with an external ring fixator in the lower extremity occurs in most cases at the ankle joint. Various treatment options are available to address tendon shortening, but accompanying physiotherapy may prevent or moderate its onset.

Improved Mobilization of Exogenous Mesenchymal Stem Cells to Bone for Fracture Healing and Sex Difference

PubMed Central

Yao, Wei; Evan Lay, Yu-An; Kot, Alexander; Liu, Ruiwu; Zhang, Hongliang; Chen, Haiyan; Lam, Kit; Lane, Nancy E.

2017-01-01

Mesenchymal stem cell (MSC) transplantation has been tested in animal and clinical fracture studies. We have developed a bone-seeking compound, LLP2A-Alendronate (LLP2A-Ale) that augments MSC homing to bone. The purpose of this study was to determine whether treatment with LLP2A-Ale or a combination of LLP2A-Ale and MSCs would accelerate bone healing in a mouse closed fracture model and if the effects are sex dependent. A right mid-femur fracture was induced in two-month-old osterix-mCherry (Osx-mCherry) male and female reporter mice. The mice were subsequently treated with placebo, LLP2A-Ale (500 µg/kg, IV), MSCs derived from wild-type female Osx-mCherry adipose tissue (ADSC, 3 × 105, IV) or ADSC + LLP2A-Ale. In phosphate buffered saline-treated mice, females had higher systemic and surface-based bone formation than males. However, male mice formed a larger callus and had higher volumetric bone mineral density and bone strength than females. LLP2A-Ale treatment increased exogenous MSC homing to the fracture gaps, enhanced incorporation of these cells into callus formation, and stimulated endochondral bone formation. Additionally, higher engraftment of exogenous MSCs in fracture gaps seemed to contribute to overall fracture healing and improved bone strength. These effects were sex-independent. There was a sex-difference in the rate of fracture healing. ADSC and LLP2A-Ale combination treatment was superior to on callus formation, which was independent of sex. Increased mobilization of exogenous MSCs to fracture sites accelerated endochondral bone formation and enhanced bone tissue regeneration. PMID:27334693

Dynamic Locked Plating of Distal Femur Fractures.

PubMed

Linn, Michael S; McAndrew, Christopher M; Prusaczyk, Beth; Brimmo, Olubusola; Ricci, William M; Gardner, Michael J

2015-10-01

Nonunion after locked bridge plating of comminuted distal femur fractures is not uncommon. "Dynamic" locked plating may create an improved mechanical environment, thereby achieving higher union rates than standard locked plating constructs. Academic Level 1 Trauma Center. Twenty-eight patients with comminuted supracondylar femur fractures treated with either dynamic or standard locked plating. Dynamic plating was achieved using an overdrilling technique of the near cortex to allow for a 0.5-mm "halo" around the screw shaft at the near cortex. Standard locked plating was done based on manufacturer's suggested technique. The patients treated with dynamic plating were matched 1:1 with those treated with standard locked plating based on OTA classification and working length. Three blinded observers made callus measurements on 6-week radiographs using a 4-point ordinal scale. The results were analyzed using a 2-tailed t test and 2-way intraclass correlations. The dynamic plating group had significantly greater callus (2.0; SD, 0.7) compared with the control group (1.3: SD, 0.8, P = 0.048) with substantial agreement amongst observers in both consistency (0.724) and absolute score (0.734). With dynamic plating group, 1 patient failed to unite, versus three in the control group (P = 0.59). The dynamic group had a mean change in coronal plane alignment of 0.5 degrees (SD, 2.6) compared with 0.6 (SD, 3.0) for the control group (P = 0.9) without fixation failure in either group. Overdrilling the near cortex in metaphyseal bridge plating can be adapted to standard implants to create a dynamic construct and increase axial motion. This technique seems to be safe and leads to increased callus formation, which may decrease nonunion rates seen with standard locked plating. Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Morphobiochemical diagnosis of acute trabecular microfractures using gamma correction Tc-99m HDP pinhole bone scan with histopathological verification.

PubMed

Bahk, Yong-Whee; Hwang, Seok-Ha; Lee, U-Young; Chung, Yong-An; Jung, Joo-Young; Jeong, Hyeonseok S

2017-11-01

We prospectively performed gamma correction pinhole bone scan (GCPBS) and histopathologic verification study to make simultaneous morphobiochemical diagnosis of trabecular microfractures (TMF) occurred in the femoral head as a part of femoral neck fracture.Materials consisted of surgical specimens of the femoral head in 6 consecutive patients. The specimens were imaged using Tc-99m hydroxymethylene diphosphonate (HDP) pinhole scan and processed by the gamma correction. After cleansing with 10% formalin solution, injured specimen surface was observed using a surgical microscope to record TMF. Morphological findings shown in the photograph, naive pinhole bone scan, GCPBS, and hematoxylin-eosin (H&E) stain of the specimen were reciprocally correlated for histological verification and the usefulness of suppression and enhancement of Tc-99m HDP uptake was biochemically investigated in TMF and edema and hemorrhage using gamma correction.On the one hand, GCPBS was able to depict the calcifying calluses in TMF with enhanced Tc-99m HDP uptake. They were pinpointed, speckled, round, ovoid, rod-like, geographic, and crushed in shape. The smallest callus measured was 0.23 mm in this series. On the other hand, GCPBS biochemically was able to discern the calluses with enhanced high Tc-99m HDP uptake from the normal and edema dipped and hemorrhage irritated trabeculae with washed out uptake.Morphobiochemically, GCPBS can clearly depict microfractures in the femoral head produced by femoral neck fracture. It discerns the microcalluses with enhanced Tc-99m HDP uptake from the intact and edema dipped and hemorrhage irritated trabeculae with suppressed washed out Tc-99m HDP uptake. Both conventional pinhole bone scan and gamma correction are useful imaging means to specifically diagnose the microcalluses naturally formed in TMF.

Comparison of microCT and an inverse finite element approach for biomechanical analysis: Results in a MSC therapeutic system for fracture healing

PubMed Central

Weis, Jared A.; Granero-Moltó, Froilán; Myers, Timothy J.; Longobardi, Lara; Spagnoli, Anna; Miga, Michael I.

2013-01-01

An important concern in the study of fracture healing is the ability to assess mechanical integrity in response to candidate therapeutics in small-animal systems. In recent reports, it has been proposed that microCT image-derived densitometric parameters could be used as a surrogate for mechanical property assessment. Recently, we have proposed an inverse methodology that iteratively reconstructs the modulus of elasticity of the lumped soft callus/hard callus region by integrating both intrinsic mechanical property (from biomechanical testing) and geometrical information (from microCT) within an inverse finite element analysis (FEA) to define a callus quality measure. In this paper, data from a therapeutic system involving mesenchymal stem cells is analyzed within the context of comparing traditional microCT densitometric and mechanical property metrics. In addition, a novel multi-parameter regression microCT parameter is analyzed as well as our inverse FEA metric. The results demonstrate that the inverse FEA approach was the only metric to successfully detect both longitudinal and therapeutic responses. While the most promising microCT-based metrics were adequate at early healing states, they failed to track late-stage mechanical integrity. In addition, our analysis added insight to the role of MSCs by demonstrating accelerated healing and was the only metric to demonstrate therapeutic benefits at late-stage healing. In conclusion, the work presented here indicates that microCT densitometric parameters are an incomplete surrogate for mechanical integrity. Additionally, our inverse FEA approach is shown to be very sensitive and may provide a first-step towards normalizing the often challenging process of assessing mechanical integrity of healing fractures. PMID:22766379

Evaluation of Fracture and Osteotomy Union in the Setting of Osteogenesis Imperfecta: Reliability of the Modified Radiographic Union Score for Tibial Fractures (RUST).

PubMed

Franzone, Jeanne M; Finkelstein, Mark S; Rogers, Kenneth J; Kruse, Richard W

2017-09-08

Evaluation of the union of osteotomies and fractures in patients with osteogenesis imperfecta (OI) is a critical component of patient care. Studies of the OI patient population have so far used varied criteria to evaluate bony union. The radiographic union score for tibial fractures (RUST), which was subsequently revised to the modified RUST, is an objective standardized method of evaluating fracture healing. We sought to evaluate the reliability of the modified RUST in the setting of the tibias of patients with OI. Tibial radiographs of 30 patients with OI fractures, or osteotomies were scored by 3 observers on 2 separate occasions. Each of the 4 cortices was given a score (1=no callus, 2=callus present, 3=bridging callus, and 4=remodeled, fracture not visible) and the modified RUST is the sum of these scores (range, 4 to 16). The interobserver and intraobserver reliabilities were evaluated using intraclass coefficients (ICC) with 95% confidence intervals. The ICC representing the interobserver reliability for the first iteration of scores was 0.926 (0.864 to 0.962) and for the second series was 0.915 (0.845 to 0.957). The ICCs representing the intraobserver reliability for each of the 3 reviewers for the measurements in series 1 and 2 were 0.860 (0.707 to 0.934), 0.994 (0.986 to 0.997), and 0.974 (0.946 to 0.988). The modified RUST has excellent interobserver and intraobserver reliability in the setting of OI despite challenges related to the poor quality of the bone and its dysplastic nature. The application and routine use of the modified RUST in the OI population will help standardize our evaluation of osteotomy and fracture healing. Level III-retrospective study of nonconsecutive patients.

Clinical Results and Complications of Lower Limb Lengthening for Fibular Hemimelia: A Report of Eight Cases.

PubMed

Mishima, Kenichi; Kitoh, Hiroshi; Iwata, Koji; Matsushita, Masaki; Nishida, Yoshihiro; Hattori, Tadashi; Ishiguro, Naoki

2016-05-01

Fibular hemimelia is a rare but the most common congenital long bone deficiency, encompassing a broad range of anomalies from isolated fibular hypoplasia up to substantial femoral and tibial shortening with ankle deformity and foot deficiency. Most cases of fibular hemimelia manifest clinically significant leg length discrepancy (LLD) with time that requires adequate correction by bone lengthening for stable walking. Bone lengthening procedures, especially those for pathological bones, are sometimes associated with severe complications, such as delayed consolidation, fractures, and deformities of the lengthened bones, leading to prolonged healing time and residual LLD at skeletal maturity. The purpose of this study was to review our clinical results of lower limb lengthening for fibular hemimelia.This study included 8 Japanese patients who diagnosed with fibular hemimelia from physical and radiological findings characteristic of fibular hemimelia and underwent single or staged femoral and/or tibial lengthening during growth or after skeletal maturity. LLD, state of the lengthened callus, and bone alignment were evaluated with full-length radiographs of the lower limb. Previous interventions, associated congenital anomalies, regenerate fractures were recorded with reference to medical charts and confirmed on appropriate radiographs. Successful lengthening was defined as the healing index <50 days/cm without regenerate fractures.A significant difference was observed in age at surgery between successful and unsuccessful lengthening. The incidence of regenerate fractures was significantly correlated with callus maturity before frame removal. LLD was corrected within 11 mm, whereas mechanical axis deviated laterally.Particular attention should be paid to the status of callus maturation and the mechanical axis deviation during the treatment period in fibular hemimelia.

The reliability of non-invasive biophysical outcome measures for evaluating normal and hyperkeratotic foot skin.

PubMed

Hashmi, Farina; Wright, Ciaran; Nester, Christopher; Lam, Sharon

2015-01-01

Hyperkeratosis of foot skin is a common skin problem affecting people of different ages. The clinical presentation of this condition can range from dry flaky skin, which can lead to fissures, to hard callused skin which is often painful and debilitating. The purpose of this study was to test the reliability of certain non-invasive skin measurement devices on foot skin in normal and hyperkeratotic states, with a view to confirming their use as quantitative outcome measures in future clinical trials. Twelve healthy adult participants with a range of foot skin conditions (xerotic skin, heel fissures and plantar calluses) were recruited to the study. Measurements of normal and hyperkeratotic skin sites were taken using the following devices: Corneometer® CM 825, Cutometer® 580 MPA, Reviscometer® RVM 600, Visioline® VL 650 Quantiride® and Visioscan® VC 98, by two investigators on two consecutive days. The intra and inter rater reliability and standard error of measurement for each device was calculated. The data revealed the majority of the devices to be reliable measurement tools for normal and hyperkeratotic foot skin (ICC values > 0.6). The surface evaluation parameters for skin: SEsc and SEsm have greater reliability compared to the SEr measure. The Cutometer® is sensitive to soft tissue movement within the probe, therefore measurement of plantar soft tissue areas should be approached with caution. Reviscometer® measures on callused skin demonstrated an unusually high degree of error. These results confirm the intra and inter rater reliability of the Corneometer®, Cutometer®, Visioline® and Visioscan® in quantifying specific foot skin biophysical properties.

Effect of strontium ranelate on fracture healing in rat tibia.

PubMed

Cebesoy, Oguz; Tutar, Ediz; Kose, Kamil Cagri; Baltaci, Yasemin; Bagci, Cahit

2007-12-01

Various anti-osteoporotic agents are available for clinical use. In contrast to other anti-osteoporotic drugs, strontium ranelate has anti-resorptive and bone-forming effects (dual action). Our objective in the present study is to investigate the efficacy of strontium ranelate (SR) on fracture healing in rat tibia. Forty-two male Wistar rats randomized into two groups (groups 1 and 2, n=21 for each). Left tibiae of all animals were broken in a closed manner using a manual three-point bending technique through mid-tibia following deep anesthesia with ketamine. The animals in group 1 were fed 25g/day specially produced food containing 450mg/kg SR starting from the first post-operative day. Group 2 were given 25g/day normal food. The animals were sacrificed on the 2nd, 3rd and 4th post-operative weeks (each week 7 animals were sacrificed from each group) and the broken tibiae were removed. The tibiae were examined first radiographically and second, histopathologically. Radiologically, callus maturity and bone union increased with time in both groups. But no significant differences were found regarding callus maturity and bone union in weekly comparisons (p=0.52, p=0.19, p=0.74). Histopathologically, it was seen that the fractures remarkably healed steadily in both groups on the 2nd, 3rd and 4th post-operative weeks. But no significant differences were found regarding the progression of fracture callus in weekly comparison (p=1.0, p=0.52, p=1.0). In the present study, we were unable to find any beneficial or harmful effects of strontium ranelate on fracture healing.

Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

USDA-ARS?s Scientific Manuscript database

Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transfe...

Influence of low-intensity laser radiation on the dynamics of some phytohormone content in the callus tissues of wheat Triticum aestivum L.

NASA Astrophysics Data System (ADS)

Dudareva, L. V.; Rudikovskaya, E. G.; Shmakov, V. N.; Rudikovskii, A. V.; Salyaev, R. K.

2017-05-01

We carried out an analysis of the influence of low-intensity He-Ne laser radiation on the dynamics of changes observed in the content of the phytohormones abscisic, jasmonic, salicylic, and indoleacetic acids in wheat calluses of Triticum aestivum L. using chromatography-mass spectrometry. He-Ne laser irradiation (λ  =  632.8 nm) in its stimulant dose (3.5 J cm-2) was proved to cause a decrease in jasmonic and indoleacetic acid content and, simultaneously, a significant (2.5-3 times) increase in ‘stress’ abscisic and salicylic acids. The response of jasmonic acid to He-Cd laser irradiation (λ  =  441 nm) was specific, which is probably due to the blue-light-induced biosynthesis of the acid. This article discusses a possible connection between substantial changes in growth regulator content within the plant tissue under the influence of low-intensity laser irradiation and the stress character of this type of radiation.

Development of guayule (Parthenium argentatum) research in cell culture

NASA Technical Reports Server (NTRS)

Ball, E. A.

1981-01-01

Utilizing the lateral buds of known high rubber producing plants as explants in culture medium specifically designed to engender shoot development and to prevent callus formation, unlimited numbers of replicate plants can be produced. Each has the same genotype as the parent. This procedure has long been used to rid plants of virus, the latter generally does not occur in the embryonic tissues of the bud; it also, by virtue of its axenic nature, eliminates all microorganisms characteristic of the parent plant. Auxins were found essential to callus formation, but since the latter is known to bring about chromosomal aberrations, it was avoided. The cytokinin benzylaminopurine strongly stimulated shoot growth, and the number of regenerated buds on the inoculum was proportional to its concentration. These buds produced shoots several centimeters in length which were caused to root on medium containing indolebutyric acid. Transferred to the septic condition of soil, the plantlets were gradually brought into full sunlight where they showed a brief vegetative growth with production of mature leaves, and flowered.

An evaluation of a new approach to the regeneration of Helichrysum italicum (Roth) G. Don, and the molecular characterization of the variation among sets of differently derived regenerants.

PubMed

Perrini, Rosaria; Alba, Vittorio; Ruta, Claudia; Morone-Fortunato, Irene; Blanco, Antonio; Montemurro, Cinzia

2009-01-01

A protocol for the induction of regeneration from leaves of Helichrysum italicum was established. Calli were found to form on the basal medium only when it was supplemented with thidiazuron (TDZ) alone or in combination with naphthalene acetic acid (NAA), with a percentage ranking of at least 80%. The hormone-free medium showed the highest percentage of shoot regeneration (62%) even though no callus formed. AFLP markers were employed to verify tissue culture-induced variation in the regenerated plantlets obtained by direct shoot regeneration or the indirect shoot regeneration process (callus formation). Seven out of the eleven AFLP primer pairs yielded polymorphic patterns. The average number of fragments per primer pair was 64.1. Singletons were represented by 12 (2.7%) fragments. Student's T-test was performed both on the average number of shared fragments and on the nucleotide diversity, and no significant statistical difference was observed between the two regeneration treatments.

Short-term exposure of Arabidopsis cell culures to hyper-G: Short-term changes in transcription regulation expression

NASA Astrophysics Data System (ADS)

Babbick, Maren; Hampp, Rudiger

2005-08-01

Callus cultures of Arabidopsis thaliana (cv. Columbia) were used to screen for early changes in gene expression in response to altered gravitational fields. In a recent microarray study we found hyper- g dependent changes in gene expression which indicated the involvement of WRKY genes [Martzivanou M. and Hampp R., Physiol. Plant., 118, 221-231,2003]. WRKY genes code for a family of plant-specific regulators of gene expression. In this study we report on the exposure of Arabidopsis callus cultures to 8g for up to 30 min. Quantitative analysis by real time RT-PCR of the amount of transcripts of WRKYs 3, 6, 22, 46, 65 and 70 showed individual changes in expression. As far as their function is known, these WRKY proteins are mainly involved in stress responses. As most alterations in transcript amount occurred within 10 min of treatment, such genes can be used for the investigation of microgravity-related effects on gene expression under sounding rocket conditions (TEXUS, MAXUS).

In Vitro Conservation of Date Palm Shoot-Tip Explants and Callus Cultures Under Minimal Growth Conditions.

PubMed

El-Dawayati, Maiada M

2017-01-01

Date palm fruit production has great economic significance for many countries. There is a fundamental necessity to conserve valuable date palm germplasm, but there are various problems with in vivo and ex situ conservation. In vitro storage has several advantages over conventional germplasm conservation methods. The in vitro technique offers a developed method of slow-growth storage, which is considered as an alternate solution for short- and medium-term storage of date palm germplasm under controlled conditions. Minimal growth conditions for germplasm conservation are generally achieved by reducing growth rate through modification of environmental growing conditions and culture, by using low temperatures, and the addition of growth retardants and osmotic agents. This chapter describes a protocol for short-term in vitro conservation of date palm shoot-tip and callus cultures under slow-growth storage conditions, using sucrose as an osmotic agent and abscisic acid (ABA) as a growth retardant at 15 °C for 12 months.

Protopine production by fumaria cell suspension cultures: effect of light.

PubMed

Georgieva, Lidiya; Ivanov, Ivan; Marchev, Andrey; Aneva, Ina; Denev, Panteley; Georgiev, Vasil; Pavlov, Atanas

2015-05-01

Protopine biosynthesis in Fumaria rostellata and Fumaria officinalis cell suspensions was investigated. For the first time, we reported for calli and cell suspensions obtained from F. rostellata and F. officinalis. Callus induction was initiated on a Murashige and Skoog medium, supplemented with sucrose and various concentrations of plant growth regulators: 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The best morphological characteristics, growth behavior, and protopine biosynthesis were observed for two callus lines (5FRL14 and 12FOL1) cultivated under submerged conditions, at low concentration of 2,4-D (0.2 and 0.5 mg/L) and higher concentration of BAP (2.0 and 3.0 mg/L). The maximal yield of protopine was accumulated from cell suspension of F. rostellata (line 5FRL14) cultivated under illumination-49.6 mg/L. Time courses of utilization of sucrose, ammonium, nitrate, and phosphate ions in cultural liquid and acetylcholinesterase inhibitory activity of alkaloid extracts of studied suspensions are also presented.

Locally applied simvastatin improves fracture healing at late period in osteoporotic rat

NASA Astrophysics Data System (ADS)

Tian, Faming; Zhang, Liu; Kang, Yuchuan; Zhang, Junshan; Ao, Jiao; Yang, Fang

effect of simvastatin locally applied from a bioactive polymer coating of implants on osteoporotic fracture healing at late period. Methods:Femur fracture model was established on normal or osteotoporotic mature female SD rats, intramedullary stabilization was achieved with uncoated titanium Kirschnerwires in normal rats(group A),with polymer-only coated vs. polymer plus simvastatin coated titanium Kirschner wires in osteoporotic rats(group B and C, respectively).Femurs were harvested after 12 weeks, and underwent radiographic and histologic analysis, as well as immunohistochemical evaluation for BMP-2 expression. Results:Radiographic results demonstrated progressed callus in the simvastatin-treated groups compared to the uncoated group.The histologic analysis revealed a significantly processed callus with irregular-shaped newly formed bone trabeculae in simvastatin-treated group. Immunohistochemical evaluation showed markedly higher expression levels of B:MP-2 in simvastatin-treated group.Conclusions: The present study revealed a improved fracture healing under local application of simvastatin in osteoporotic rat,which might partially from upregulation of the B:MP-2 expression at fractured site.

MiR-142-5p promotes bone repair by maintaining osteoblast activity.

PubMed

Tu, Manli; Tang, Juanjuan; He, Hongbo; Cheng, Peng; Chen, Chao

2017-05-01

MicroRNAs play important roles in regulating bone regeneration and remodeling. However, the pathophysiological roles of microRNAs in bone repair remain unclear. Here we identify a significant upregulation of miR-142-5p correlated with active osteoblastogenesis during the bone healing process. In vitro, miR-142-5p promoted osteoblast activity and matrix mineralization by targeting the gene encoding WW-domain-containing E3 ubiquitin protein ligase 1. We also found that the expression of miR-142-5p in the callus of aged mice was lower than that in the callus of young mice and directly correlated with the age-related delay in bone healing. Furthermore, treatment with agomir-142-5p in the fracture areas stimulated osteoblast activity which repaired the bone fractures in aged mice. Thus, our study revealed that miR-142-5p plays a crucial role in healing fractures by maintaining osteoblast activity, and provided a new molecular target therapeutic strategy for bone healing.

Effects of elicitors on the production of resveratrol and viniferins in cell cultures of Vitis vinifera L. cv Italia.

PubMed

Santamaria, Anna Rita; Mulinacci, Nadia; Valletta, Alessio; Innocenti, Marzia; Pasqua, Gabriella

2011-09-14

Methyl jasmonate, jasmonic acid and chitosan were tested as elicitors on cell suspension cultures obtained from Vitis vinifera cv Italia to investigate their effect on stilbene production. Stilbene accumulation in the callus, grown under nonelicited conditions, was also investigated. Calli and cell suspensions were obtained in a B5 culture medium supplemented with 0.2 mg L(-1) NAA and 1 mg L(-1) KIN. Stilbene determination was achieved by HPLC/DAD/MS. Whereas callus biosynthesized only piceid, cell suspensions elicited with jasmonates produced several stilbenes, mainly viniferins. In suspended cells, methyl jasmonate and jasmonic acid were the most effective in stimulating stilbene biosynthesis, whereas chitosan was less effective; in fact, the amount of stilbenes obtained with this elicitor was not significantly different from that obtained for the control cells. The maximum production of total stilbenes was at day 20 of culture with 0.970 and 1.023 mg g(-1) DW for MeJA and JA, respectively.

Bone bonding through bioadhesives: present status.

PubMed

Meyer, G; Muster, D; Schmitt, D; Jung, P; Jaeger, J H

1979-01-01

Until recently use of adhesives was confined to cases in which glued areas could be pre-treated or at least cleaned. Thus, grease or oil contaminated surfaces could not be joined together by glueing. More recently, some adhesives have been developed which allow previous treatment of greasy surfaces to be avoided. Among these we find epoxy resins, acrylics and polyurethances. These adhesives have been used until now in various industries. We have begun a research program with these products and in aiming to design an adhesive which would enable immediate and strong bone bonding and avoid problems of metallic fixation, this study is a continuation of our previous research. Thus we tested - currently available surgical and dental adhesives - original mixtures developed in our laboratory. Mechanical assays were performed on bone samples from human femurs in different conditions : dried, cleaned, fresh, or after immersion in physiological solution. They consist essentially of tensile tests on Lhomargy and Zwick's machine wherein the stress is directed perpendicular to the interface. Variations of tensile strength (in h bar) are related to hardening time and to mixture composition. The specimens are joined together either in monolayers or in multilayers. The use of adequate catalysts ensures setting at room temperature. Torsion tests and fatigue tests are carried out concomitantly. Standardized bevel fermoral osteotomies were performed on mice with a dental saw after I.P. Nembutal anesthesia in order to test biological tolerance : - for the control group we study the evolution of bone repair after circumferential wiring - for the animals under test, bones are glued together with one of the proposed adhesives. Radiological and histological studies (using classical Azantrichrome staining after demineralization) are carried out at regular time intervals. In the control animals particular attention is paid to the time course of the formation, constitution and evolution of callus. In the test animals, we can observe callus formation, bone growth into the adhesive material and glue resorption, and look for specific antigenic phenomena. Despite expected improvements, bone glueing remains a challenge and only restricted clinical applications can be proposed.

Endochondral Ossification for Enhancing Bone Regeneration: Converging Native Extracellular Matrix Biomaterials and Developmental Engineering In Vivo

PubMed Central

Dennis, S. Connor; Berkland, Cory J.; Bonewald, Lynda F.

2015-01-01

Autologous bone grafting (ABG) remains entrenched as the gold standard of treatment in bone regenerative surgery. Consequently, many marginally successful bone tissue engineering strategies have focused on mimicking portions of ABG's “ideal” osteoconductive, osteoinductive, and osteogenic composition resembling the late reparative stage extracellular matrix (ECM) in bone fracture repair, also known as the “hard” or “bony” callus. An alternative, less common approach that has emerged in the last decade harnesses endochondral (EC) ossification through developmental engineering principles, which acknowledges that the molecular and cellular mechanisms involved in developmental skeletogenesis, specifically EC ossification, are closely paralleled during native bone healing. EC ossification naturally occurs during the majority of bone fractures and, thus, can potentially be utilized to enhance bone regeneration for nearly any orthopedic indication, especially in avascular critical-sized defects where hypoxic conditions favor initial chondrogenesis instead of direct intramembranous ossification. The body's native EC ossification response, however, is not capable of regenerating critical-sized defects without intervention. We propose that an underexplored potential exists to regenerate bone through the native EC ossification response by utilizing strategies which mimic the initial inflammatory or fibrocartilaginous ECM (i.e., “pro-” or “soft” callus) observed in the early reparative stage of bone fracture repair. To date, the majority of strategies utilizing this approach rely on clinically burdensome in vitro cell expansion protocols. This review will focus on the confluence of two evolving areas, (1) native ECM biomaterials and (2) developmental engineering, which will attempt to overcome the technical, business, and regulatory challenges that persist in the area of bone regeneration. Significant attention will be given to native “raw” materials and ECM-based designs that provide necessary osteo- and chondro-conductive and inductive features for enhancing EC ossification. In addition, critical perspectives on existing stem cell-based therapeutic strategies will be discussed with a focus on their use as an extension of the acellular ECM-based designs for specific clinical indications. Within this framework, a novel realm of unexplored design strategies for bone tissue engineering will be introduced into the collective consciousness of the regenerative medicine field. PMID:25336144

Polycaprolactone/glass bioabsorbable implant in a rabbit humerus fracture model.

PubMed

Lowry, K J; Hamson, K R; Bear, L; Peng, Y B; Calaluce, R; Evans, M L; Anglen, J O; Allen, W C

1997-09-15

Research in improved materials and methods for internal fixation has centered on internal fixators made of bioabsorbable materials such as polylactic acid, polyglycolic acid, and polyparadioxanone. These materials have two problems: the first is a postoperative complication related to a delayed inflammatory response; and the second is low strength characteristics. An alternative material developed to alleviate these problems is a composite of phosphate glass fibers embedded in the polymer polycaprolactone, referred to as PCL. In this study, intramedullary pins made of PCL were compared to stainless steel pins in a rabbit humerus osteotomy model. Specimens were harvested at 0, 6, and 12 weeks postoperatively, radiographs and mechanical testing to failure were performed at each time interval, and tissue was examined microscopically at 6 and 12 weeks. Histologic results showed PCL pins to be well tolerated with minimal inflammation around the pin. Mechanical testing revealed the PCL fixation to be weaker initially than the stainless steel fixation. There was significant stress shielding of stainless-steel-healed rabbit humeri when compared to the PCL/bone humeri. All osteotomies immobilized with PCL healed with abundant periosteal callus production.

Chitosan stabilizes platelet growth factors and modulates stem cell differentiation toward tissue regeneration.

PubMed

Busilacchi, Alberto; Gigante, Antonio; Mattioli-Belmonte, Monica; Manzotti, Sandra; Muzzarelli, Riccardo A A

2013-10-15

The idea of using chitosan as a functional delivery aid to support simultaneously PRP, stem cells and growth factors (GF) is associated with the intention to use morphogenic biomaterials to modulate the natural healing sequence in bone and other tissues. For example, chitosan-chondroitin sulfate loaded with platelet lysate was included in a poly(D,L-lactate) foam that was then seeded with human adipose-derived stem cells and cultured in vitro under osteogenic stimulus: the platelet lysate provided to the bone tissue the most suitable assortment of GF which induces the osteogenic differentiation of the mesenchymal stem cells. PDGF, FGF, IGF and TGF-β were protagonists in the repair of callus fractures. The release of GF from the composites of chitosan-PRP and either nano-hydroxyapatite or tricalcium phosphate was highly beneficial for enhancing MSC proliferation and differentiation, thus qualifying chitosan as an excellent vehicle. A number of biochemical characteristics of chitosan exert synergism with stem cells in the regeneration of soft tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

Demonstrations at School Level of the Effects of IAA

ERIC Educational Resources Information Center

Falk, Peter

1973-01-01

Describes demonstrations suitable for secondary school biology classes relating to the effects of the hormone IAA on plant growth. Demonstrations illustrate how hormone treatments affect stem elongation, callus formation, inhibition of axillary buds, stimulation of secondary growth and initiation of adventitious root development, root elongation,…

Timing of Ethylene Modification Is Critical For Regeneration In Barley

USDA-ARS?s Scientific Manuscript database

: The plant hormone ethylene is important for higher rates of callus formation and green plant regeneration. Ethylene can have positive or negative effects on these traits depending on the genotype, type of explant and stage of application. Therefore, the effects of both ethylene precur...

EFFECTS OF MEDIUM COMPONENTS AND LIGHT ON CALLUS INDUCTION, GROWTH, AND FROND REGENERATION IN LEMNA GIBBA (DUCKWEED). (R823570)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Physiological Challenges of Bone Repair

DTIC Science & Technology

2012-12-01

expression, in general, followed the same pattern in both groups, but significantly, lower levels of mRNA for Indian Hedgehog (ihh) and BMP-2 were detected in...the fracture calluses of the older rats. Indian Hedgehog is thought to be involved in chondrogenesis and bone repair, whereas BMP-2 stimulates bone

Inoculation methods for Populus tremuloides resistant to Hypoxylon canker

Treesearch

S. A. Enebak; Michael E. Ostry; N. A. Anderson

1999-01-01

Canker expansion and the amount of callus tissue formed were measured monthly on 60 ramets from each of five trembling aspen (Populus tremuloides Michx.) clones that had been inoculated in wounds with Entoleuca mammata (= Hypoxylon marnmatum (Wahl.) Mill) over a 12-month period. At the clone level, the prevalence...

29 CFR 776.20 - “Goods.”

Code of Federal Regulations, 2013 CFR

2013-07-01

..., bills of lading, checks, drafts, negotiable notes and other commercial paper. 26 “Goods” includes gold... or rented; 33 ice; 34 containers, as, for example, cigar boxes or wrapping paper and packing... of the four dissenting justices in 10 E. 40th St. Bldg. v. Callus, 325 U.S. at p. 586. Waste paper...

29 CFR 776.20 - “Goods.”

Code of Federal Regulations, 2014 CFR

2014-07-01

..., bills of lading, checks, drafts, negotiable notes and other commercial paper. 26 “Goods” includes gold... or rented; 33 ice; 34 containers, as, for example, cigar boxes or wrapping paper and packing... of the four dissenting justices in 10 E. 40th St. Bldg. v. Callus, 325 U.S. at p. 586. Waste paper...

29 CFR 776.20 - “Goods.”

Code of Federal Regulations, 2012 CFR

2012-07-01

..., bills of lading, checks, drafts, negotiable notes and other commercial paper. 26 “Goods” includes gold... or rented; 33 ice; 34 containers, as, for example, cigar boxes or wrapping paper and packing... of the four dissenting justices in 10 E. 40th St. Bldg. v. Callus, 325 U.S. at p. 586. Waste paper...

Optimized conditions for biolistic-mediated transformation of Lilium longilforum 'Nellie White'

USDA-ARS?s Scientific Manuscript database

A variety of tissues were used for biolistic-mediated transformation of Lilum longiflorum 'Nellie White'. Transgenic plants were not recovered from five-month-old, non-embryogenic callus or suspension cells that had been bombarded with pDM327 that contains the bar-uidA fusion gene under control the ...

In Vivo Performance of Bilayer Hydroxyapatite Scaffolds for Bone Tissue Regeneration in the Rabbit Radius

DTIC Science & Technology

2011-02-02

no treatments and the pres- ence of periosteal callus-like layer surrounding defects with scaffold implantation were observed after 8 weeks post...vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model. Biomaterials. 2004; 25(20):5037–44. 20. Lu JX, Gallur A, Flautre

Clonal variation in lateral and basal rooting of Populus irrigated with landfill leachate

Treesearch

R.S. Zalesny Jr.; J.A. Zalesny

2011-01-01

Successful establishment and productivity of Populus depends upon adventitious rooting from: 1) lateral roots that develop from either preformed or induced primordia and 2) basal roots that differentiate from callus at the base of the cutting in response to wounding. Information is needed for phytotechnologies about the degree to which ...

Civilian Health and Medical Program of the Uniformed Services (CHAMPUS). Change 5

DTIC Science & Technology

1993-09-17

exclusion. 4-31c b. Limitations. 4-32 7. Transsexualism or hemapbroditim. 4-32 a. Cosmetic, reconstructive, or platic surgery. 4-32 a. Limited benefits...Obesity, weight reduction. 4-49 29. Transsexualism or hermaphroditism. 4-49 30. Sexual dysfunctions or inadequacies. 4-49 31. Corns, calluses, and toenails

Civilian Health and Medical Program of the Uniformed Services (CHAMPUS). Change 6

DTIC Science & Technology

1994-06-24

Exception to general exclusion. 4-31c b. Limitations. 4-32 7. Transsexualism or hermaphroditism. 4-32 8. Cosmetic, reconstructive, or plastic surgery. 4... Transsexualism or hermaphroditism. 4-49 30. Sexual dysfunctions or inadequacies. 4-49 31. Corns, calluses, and toenails. 4-49 32. Dyslexia. 4-49 33

Using Haworthia Cultured Cells as an Aid in Teaching Botany

ERIC Educational Resources Information Center

Majumdar, Shyamal K.; Castellano, John M.

1977-01-01

Callus induction from species of Haworthia can be done quickly in the laboratory with minimal equipment to study tissue dedifferentiation and cellular redifferentiation. It is shown that the cultured cell can also be used to study and evaluate the effects of various mutagens, carcinogens, and pesticides in controlled environments. (Author/MA)

Efficient in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood) and assessment of genetic fidelity of micropropagated plants.

PubMed

Shinde, Smita; Sebastian, Joseph Kadanthottu; Jain, Jyothi Ramesh; Hanamanthagouda, Manohar Shirugumbi; Murthy, Hosakatte Niranjana

2016-10-01

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon.

PubMed

Yamamura, Yoshimi; Sahin, F Pinar; Nagatsu, Akito; Mizukami, Hajime

2003-04-01

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.

Responses to cadmium toxicity during in vitro growth in Arachis hypogaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakravarty, B.; Srivastava, S.

1994-05-01

Toxic effects of increasing concentration of cadmium (Cd) in the environment due to rapid industrialization has become a major environmental concern. Cadmium is readily taken up by the plant system which retards the growth and yield of crops. It has been implicated inhibition of the photosynthetic mechanism as well as nodulation, nitrogen fixation, and consequently the growth of leguminous plants. Cell cultures are convenient systems for the study of mechanism of metal toxicity, as they eliminate the interfering processes of translocation and organ-specific trapping of metal ions. Genetic manipulations for metal tolerance can improve the responsiveness to the environmental stressmore » and promote productivity of plants. To determine the extent of tolerance and raise resistant callus lines, cultures of Arachic hypogaea (groundnut), an important oil-yielding crop, were subjected to different concentrations of Cd in the media. The callus growth during the different passages as well as the percentage of dividing cells and mitotic abnormalities was recorded. Also, the protein profiles, quantitative and qualitative, were studied to detect whether synthesis of Cd-binding protein occurs.« less

Early somatic embryo induction events in alfalfa callus cultures. [Medicago sativa

DOE Office of Scientific and Technical Information (OSTI.GOV)

El-Bakry, A.A.; Hildebrand, D.F.

1987-04-01

High and low regenerating alfalfa Medicago sativa L. cv Regen S full sibs were isolated from a callus culture screen on modified Blaydes medium. The average number of embryos per ovary were thirty and zero for the high and low genotypes respectively after six weeks in culture. Proembryonic cell masses (4-8 celled) were observed after 4-5 days in culture and maximum meristematic activity was at 6-7 days in culture, for the high regenerating genotypes. Well formed globular embryos, both epidermal and subepidermal in origin, were observed after 2 weeks is culture. Samples in culture for 3, 6 and 14 daysmore » from the high and low regenerating genotypes were radiolabeled in vivo with /sup 35/S-methionine and run both on one and two dimension gels. The results will be discussed in relation to differences in proteins between the high and low regenerating genotypes at the stage of maximum meristematic activity (day 6) and differences occurring relative to the appearance of globular stage embryos (day 14) will be presented.« less

Cell and tissue culture of Miscanthus Sacchariflorus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Godovikova, V.A.; Moiseyeva, E.A.; Shumny, V.K.

1995-11-01

Since recent time search and introduction of new species of plants have paid attention. More perspective are perennial low maintenance landscape plants from genera Phragmites L. and Miscanthus Anderss. known as high speed growing and great amount of cellulose`s containing. Absence of seeds production and limited distribution area prevent from immediately introduction the plants of this species. The main goal of our investigation is the scientific development of the cell and tissue culture methods to get changing clones, salt and cold tolerant plants and their micropogation. At present there are collection of biovariety represented by subspecies, ecotypes and plant regenerantsmore » of two species - Miscanthus purpurascens (Anders.) and Miscanthus sacchariflorus (Maxim.). Successful results have been achieved in screening of culture media, prepared on MS base medium and contained a row of tropic components to protect the explant and callus tissue from oxidation and necrosis. Initially the callus was induced from stem segments, apical and nodular meristem of vegetative shoots of elulalia, growing in hydroponic greenhouse. Morphological and cytologic analysis of plant-regenerants have been done.« less

Stress fractures of the ribs in elite competitive rowers: a report of nine cases.

PubMed

Dragoni, S; Giombini, A; Di Cesare, A; Ripani, M; Magliani, G

2007-10-01

The objective was to report the clinical and imaging patterns of nine cases of stress fractures of the rib diagnosed in Italian Olympic rowers. Nine patients with stress fractures of the rib detected from 103 (8.7%) Italian team rowers competing between May 2000 and May 2006 were identified based on the database of a sports medicine institute. All athletes were male. They were aged between 17 and 31 years (mean: 24.4). Patient weight, fracture location, rowing side and imaging methods employed were noted. The diagnosis was made based on history, clinical examination and Tc-99m MDP bone scintigraphy. Eight of the 9 fractures were located anterolaterally between the fourth and ninth rib. In 1 of the 5 athletes with standard radiographs, a fracture line was visible and in 4 there was callus formation. In 2 athletes sonography was performed, which detected discontinuity of the rib surface and callus formation (1 case each). Stress fractures of the ribs are relatively common in competitive rowers. They are characterized by increasing lateral chest pain and typical scintigraphic, radiographic and sonographic findings.

[Genetic mutation and clinical features of osteogenesis imperfecta type V].

PubMed

Guan, Shizhen; Bai, Xue; Wang, Yi; Liu, Zhigang; Ren, Xiuzhi; Zhang, Tianke; Ju, Mingyan; Li, Keqiu; Li, Guang

2017-12-10

To explore genetic mutations and clinical features of osteogenesis imperfecta type V. Clinical record of five patients (including one familial case) with osteogenesis imperfecta type V were retrospectively analyzed. Peripheral blood samples of the patients, one family member, as well as healthy controls were collected. Mutation of IFITM5 gene was identified by PCR amplification and Sanger sequencing. A heterozygous mutation (c.-14C>T) in the 5-UTR of the IFITM5 gene was identified in all of the patients and one mother. The clinical findings included frequent fractures and spine and/or extremities deformities, absence of dentinogenesis imperfecta, absence of hearing impairment, and blue sclera in 1 case. Radiographic findings revealed calcification of the interosseous membrane between the radius-ulna in all cases. Hyperplastic callus formation was found in 3 cases. Four had radial-head dislocation. A single heterozygous mutation c.-14C>T was found in the 5-UTR of the IFITM5 gene in 5 patients with osteogensis imperfecta type V. The patients showed specific radiological features including calcification of interosseous membrane, hyperplastic callus formation, and radial-head dislocation.

Unfertilized ovary: a novel explant for coconut (Cocos nucifera L.) somatic embryogenesis.

PubMed

Perera, Prasanthi I P; Hocher, Valerie; Verdeil, Jean Luc; Doulbeau, Sylvie; Yakandawala, Deepthi M D; Weerakoon, L Kaushalya

2007-01-01

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 microM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 microM abscisic acid, followed by plant regeneration medium (with 5 microM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.

Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

NASA Technical Reports Server (NTRS)

Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

2000-01-01

Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

Common foot problems in diabetic foot clinic.

PubMed

Tantisiriwat, Natthiya; Janchai, Siriporn

2008-07-01

To study common foot problems presented in diabetic foot clinic. A retrospectively review of out patient department records and diabetic foot evaluation forms of patients who visited the diabetic foot clinic at King Chulalongkorn Memorial Hospital between 2004 and 2006. Of all diabetic patients, 70 men and 80 women with the average age of 63.8 years were included in this study. About 32% of all reported cases had lower extremity amputation in which the toe was the most common level. Foot problems were evaluated and categorized in four aspects, dermatological, neurological, musculoskeletal, and vascular, which were 67.30%, 79.3%, 74.0%, and 39.3% respectively. More than half of the patients had skin dryness, nail problem and callus formation. Fifty six percent had the abnormal plantar pressure area, which was presented as callus. The great toe was the most common site of callus formation, which was correlated with gait cycle. The current ulcer was 18.8%, which was presented mostly at heel and great toe. Three-fourth of the patients (75.3%) had lost protective sensation, measured by the 5.07 monofilament testing. The most common problem found in musculoskeletal system was limited motion of the joint (44.0%). Claw toe or hammer toe were reported as 32.0% whereas the other deformities were bunnion (12.0%), charcot joint (6.0%) and flat feet (5.3%). The authors classified patients based on category risk to further lower extremity amputation into four groups. Forty-seven percent had highest risk for having further amputation because they had lost protective sensation from monofilament testing, previous current ulcer, or history of amputation. Only half of the patients had previous foot care education. Multidisciplinary diabetic foot care including patient education (proper foot care and footwear), early detection, effective management of foot problems, and scheduled follow-up must be emphasized to prevent diabetes-related lower extremities amputation.

Effect of different in vitro culture extracts of black pepper (Piper nigrum L.) on toxic metabolites-producing strains.

PubMed

Ahmad, Nisar; Abbasi, Bilal Haider; Fazal, Hina

2016-03-01

In the present study, the effect of different in vitro cultures (callus, in vitro shoots) and commercially available peppercorn extract was investigated for its activity against toxic metabolite-producing strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Candida albicans). These in vitro cultures were extracted with ethanol, hexane, and chloroform, and the antipathogenic activity was determined by well-diffusion method. Hexane extract of callus showed 22 mm zone of inhibition against B. cereus, 23 mm against S. aureus, while regenerated shoots and seeds have shown 24.3 and 26 mm zones of inhibition. The ethanolic extracts of regenerated Piper shoots have shown 25 mm activity against S. aureus, 21 mm against B. cereus, and 16 mm in the case of C. albicans in comparison with standard antibiotics. Peppercorn extracts in chloroform and ethanol had shown activities against B. cereus (23.6 mm) and B. subtilis (23.5 mm). During in vitro organogenesis and morphogenesis, cells and tissues produced a comparable phytochemicals profile like mother plant. Morphogenesis is critically controlled by the application of exogenous plant-growth regulators. Such addition alters the hormonal transduction pathways, and cells under in vitro conditions regenerate tissues, which are dependant on the physiological state of cells, and finally enhance the production of secondary metabolites. To the best of our knowledge, this is the first report to compare the antimicrobial potential of in vitro regenerated tissues and peppercorn with standard antibiotics. In conclusion, most of the extracts showed pronounced activities against all the pathogenic microbes. This is a preliminary work, and the minimum inhibitory concentration values needs to be further explored. Regenerated tissues of P. nigrum are a good source of biologically active metabolites for antimicrobial activities, and callus culture presented itself as a good candidate for such activities. © The Author(s) 2013.

A study on the influence of different promoter and 5'UTR (URM) cassettes from Arabidopsis thaliana on the expression level of the reporter gene β glucuronidase in tobacco and cotton.

PubMed

Agarwal, Parul; Garg, Varsha; Gautam, Taru; Pillai, Beena; Kanoria, Shaveta; Burma, Pradeep Kumar

2014-04-01

Several reports of promoters from plants, viral and artificial origin that confer high constitutive expression are known. Among these the CaMV 35S promoter is used extensively for transgene expression in plants. We identified candidate promoters from Arabidopsis based on their transcript levels (meta-analysis of available microarray control datasets) to test their activity in comparison to the CaMV 35S promoter. A set of 11 candidate genes were identified which showed high transcript levels in the aerial tissue (i.e. leaf, shoot, flower and stem). In the initial part of the study binary vectors were developed wherein the promoter and 5'UTR region of these candidate genes (Upstream Regulatory Module, URM) were cloned upstream to the reporter gene β glucuronidase (gus). The promoter strengths were tested in transformed callus of Nicotiana tabacum and Gossypium hirsutum. On the basis of the results obtained from the callus, the influence of the URM cassettes on transgene expression was tested in transgenic tobacco. The URM regions of the genes encoding a subunit of photosystem I (PHOTO) and geranyl geranyl reductase (GGR) in A. thaliana genome showed significantly high levels of GUS activity in comparison to the CaMV 35S promoter. Further, when the 5'UTRs of both the genes were placed downstream to the CaMV 35S promoter it led to a substantial increase in GUS activity in transgenic tobacco lines and cotton callus. The enhancement observed was even higher to that observed with the viral leader sequences like Ω and AMV, known translational enhancers. Our results indicate that the two URM cassettes or the 5'UTR regions of PHOTO and GGR when placed downstream to the CaMV 35S promoter can be used to drive high levels of transgene expression in dicotyledons.

Systemic Delivery of Free Chitosan Accelerates Femur Fracture Healing in Rats.

PubMed

Shao, Peng; Wei, Yongzhong; Dass, Crispin R; Zhang, Guoying; Wu, Zhisheng

2018-01-01

Chitosan-containing compounds have been shown to be suitable for bone replacement, but few studies demonstrate the impact of the chitosan as a free drug on the fracture.In this study, we aimed to evaluate possible effects of free chitosan on fracture healing. Thirty adult male Sprague-Dawley rats with a mean body weight of 205 g (range from 200g to 210g) were randomly and equally divided into two groups. Standardized femur fractures were created in all rats. Treatments were administered intraperitoneally twice weekly at 1 mg chitosan per injection and the controls were administered physiological saline. The site of the fracture was compared with the control group at 1, 2 and 4 weeks after surgery (n=5 in each group). The weight, activity and reaction of the rats were observed at all the timepoints. Anterior-posterior radiographs and micro-CT scans of all fractures were taken after surgery, and the parameters included: the volume of callus that was calculated using the Perkins volume formula, BV/TV, BV, BMD of cortical bone, cortical thickness, and cortical number at the fracture sites. After sacrifice, fractured femurs from rats were dissected and carefully cleaned of muscle around the fracture callus to preserve callus integrity. Sections were stained with haematoxylin and eosin for histological evaluation of healing. Radiological (X-ray and micro-CT) evaluation showed that fracture healing of the experimental group was better than control group at the second week and fourth week. Histological evaluation revealed fracture healing of the experimental group was better than control group at the same time. There was no statistically significant difference in fracture healing between the two groups at the first week. Systemic delivery of free chitosan can accelerate the bone healing process in rat femur fracture at the early-middle stage. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes

PubMed Central

Bestoso, Federica; Ottaggio, Laura; Armirotti, Andrea; Balbi, Alessandro; Damonte, Gianluca; Degan, Paolo; Mazzei, Mauro; Cavalli, Francesca; Ledda, Bernardetta; Miele, Mariangela

2006-01-01

Background Taxol is an effective antineoplastic agent, originally extracted from the bark of Taxus brevifolia with a low yield. Many attempts have been made to produce Taxol by chemical synthesis, semi-synthesis and plant tissue cultures. However, to date, the availability of this compound is not sufficient to satisfy the commercial requirements. The aim of the present work was to produce suspension cell cultures from plants not belonging to Taxus genus and to verify whether they produced Taxol and taxanes. For this purpose different explants of hazel (Corylus avellana species) were used to optimize the protocol for inducing in vitro callus, an undifferentiated tissue from which suspension cell cultures were established. Results Calli were successfully induced from stems, leaves and seeds grown in various hormone concentrations and combinations. The most suitable callus to establish suspension cell cultures was obtained from seeds. Media recovered from suspension cell cultures contained taxanes, and showed antiproliferative activity on human tumour cells. Taxol, 10-deacetyltaxol and 10-deacetylbaccatin III were the main taxanes identified. The level of Taxol recovered from the media of hazel cultures was similar to that found in yew cultures. Moreover, the production of taxanes in hazel cell cultures increased when elicitors were used. Conclusion Here we show that hazel cell cultures produce Taxol and taxanes under controlled conditions. This result suggests that hazel possesses the enzymes for Taxol production, which until now was considered to be a pathway particular to Taxus genus. The main benefit of producing taxanes through hazel cell cultures is that hazel is widely available, grows at a much faster rate in vivo, and is easier to cultivate in vitro than yew. In addition, the production of callus directly from hazel seeds shortens the culture time and minimizes the probability of contamination. Therefore, hazel could become a commercial source of Taxol and taxanes, both to be used as new therapeutic agents or as new precursors for Taxol semi-synthesis. PMID:17150090

Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing.

PubMed

Wang, Liping; Hsiao, Edward C; Lieu, Shirley; Scott, Mark; O'Carroll, Dylan; Urrutia, Ashley; Conklin, Bruce R; Colnot, Celine; Nissenson, Robert A

2015-10-01

G-protein-coupled receptors (GPCRs) are key regulators of skeletal homeostasis and are likely important in fracture healing. Because GPCRs can activate multiple signaling pathways simultaneously, we used targeted disruption of G(i) -GPCR or activation of G(s) -GPCR pathways to test how each pathway functions in the skeleton. We previously demonstrated that blockade of G(i) signaling by pertussis toxin (PTX) transgene expression in maturing osteoblastic cells enhanced cortical and trabecular bone formation and prevented age-related bone loss in female mice. In addition, activation of G(s) signaling by expressing the G(s) -coupled engineered receptor Rs1 in maturing osteoblastic cells induced massive trabecular bone formation but cortical bone loss. Here, we test our hypothesis that the G(i) and G(s) pathways also have distinct functions in fracture repair. We applied closed, nonstabilized tibial fractures to mice in which endogenous G(i) signaling was inhibited by PTX, or to mice with activated G(s) signaling mediated by Rs1. Blockade of endogenous G(i) resulted in a smaller callus but increased bone formation in both young and old mice. PTX treatment decreased expression of Dkk1 and increased Lef1 mRNAs during fracture healing, suggesting a role for endogenous G(i) signaling in maintaining Dkk1 expression and suppressing Wnt signaling. In contrast, adult mice with activated Gs signaling showed a slight increase in the initial callus size with increased callus bone formation. These results show that G(i) blockade and G(s) activation of the same osteoblastic lineage cell can induce different biological responses during fracture healing. Our findings also show that manipulating the GPCR/cAMP signaling pathway by selective timing of G(s) and G(i) -GPCR activation may be important for optimizing fracture repair. © 2015 American Society for Bone and Mineral Research.

The Effect of Vibration Treatments Combined with Teriparatide or Strontium Ranelate on Bone Healing and Muscle in Ovariectomized Rats.

PubMed

Komrakova, M; Hoffmann, D B; Nuehnen, V; Stueber, H; Wassmann, M; Wicke, M; Tezval, M; Stuermer, K M; Sehmisch, S

2016-10-01

The aim of the present study was to study the effect of combined therapy of teriparatide (PTH) or strontium ranelate (SR) with whole-body vibration (WBV) on bone healing and muscle properties in an osteopenic rat model. Seventy-two rats (3 months old) were bilaterally ovariectomized (Ovx), and 12 rats were left intact (Non-Ovx). After 8 weeks, bilateral transverse osteotomy was performed at the tibia metaphysis in all rats. Thereafter, Ovx rats were divided into six groups (n = 12): (1) Ovx-no treatment, (2) Ovx + vibration (Vib), (3) SR, (4) SR + Vib, (5) PTH, and (6) PTH + Vib. PTH (40 μg/kg BW sc. 5×/week) and SR (613 mg/kg BW in food daily) were applied on the day of ovariectomy, vibration treatments 5 days later (vertical, 70 Hz, 0.5 mm, 2×/day for 15 min) for up to 6 weeks. In the WBV + SR group, the callus density, trabecular number, and Alp and Oc gene expression were decreased compared to SR alone. In the WBV + PTH group, the cortical and callus widths, biomechanical properties, Opg gene expression, and Opg/Rankl ratio were increased; the cortical and callus densities were decreased compared to PTH alone. A case of non-bridging was found in both vibrated groups. Vibration alone did not change the bone parameters; PTH possessed a stronger effect than SR therapy. In muscles, combined therapies improved the fiber size of Ovx rats. WBV could be applied alone or in combination with anti-osteoporosis drug therapy to improve muscle tissue. However, in patients with fractures, anti-osteoporosis treatments and the application of vibration could have an adverse effect on bone healing.

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2).

PubMed

Ganesan, M; Jayabalan, N

2004-10-01

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH4NO3 and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6-7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.

High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction, and unsuspected persistence.

PubMed

Thomas, Pious; Sekhar, Aparna C; Shaik, Sadiq Pasha

2017-11-01

Molecular and microscopic analyses reveal enormous non-cultivable endophytic bacteria in grapevine field shoots with functional significance. Diverse bacteria enter tissue cultures through surface-sterilized tissues and survive surreptitiously with varying taxonomic realignments. The study was envisaged to assess the extent of endophytic bacterial association with field shoot tissues of grapevine and the likelihood of introduction of such internally colonizing bacteria in vitro adopting molecular techniques targeting the non-cultivable bacterial community. PowerFood ® -kit derived DNA from surface-sterilized field shoot tips of grapevine Flame Seedless was employed in a preliminary bacterial class-specific PCR screening proving positive for major prokaryotic taxa including Archaea. Taxonomic and functional diversity were analyzed through whole metagenome profiling (WMG) which revealed predominantly phylum Actinobacteria, Proteobacteria, and minor shares of Firmicutes, Bacteroidetes, and Deinococcus-Thermus with varying functional roles ascribable to the whole bacterial community. Field shoot tip tissues and callus derived from stem segments were further employed in 16S rRNA V3-V4 amplicon taxonomic profiling. This revealed elevated taxonomic diversity in field shoots over WMG, predominantly Proteobacteria succeeded by Actinobacteria, Firmicutes, Bacteroidetes, and 15 other phyla including several candidate phyla (135 families, 179 genera). Callus stocks also displayed broad bacterial diversity (16 phyla; 96 families; 141 genera) bearing resemblance to field tissues with Proteobacterial dominance but a reduction in its share, enrichment of Actinobacteria and Firmicutes, disappearance of some field-associated phyla and detection of a few additional taxonomic groups over field community. Similar results were documented during 16S V3-V4 amplicon taxonomic profiling on Thompson Seedless field shoot tip and callus tissues. Video microscopy on tissue homogenates corroborated enormous endophytic bacteria. This study elucidates a vast diversity of cultivation-recalcitrant endophytic bacteria prevailing in grapevine field shoots, their in vitro introduction, and unsuspecting sustenance with possible silent participation in tissue culture processes.

The Phaseolus vulgaris PvTRX1h gene regulates plant hormone biosynthesis in embryogenic callus from common bean.

PubMed

Barraza, Aarón; Cabrera-Ponce, José L; Gamboa-Becerra, Roberto; Luna-Martínez, Francisco; Winkler, Robert; Álvarez-Venegas, Raúl

2015-01-01

Common bean is the most important grain legume in the human diet. Bean improvement efforts have been focused on classical breeding techniques because bean is recalcitrant to both somatic embryogenesis and in vitro regeneration. This study was undertaken to better understand the process of somatic embryogenesis in the common bean. We focused on the mechanisms by which somatic embryogenesis in plants is regulated and the interaction of these mechanisms with plant hormones. Specifically, we examined the role of the gene PvTRX1h, an ortholog of a major known histone lysine methyltransferase in plants, in somatic embryo generation. Given the problems with regeneration and transformation, we chose to develop and use regeneration-competent callus that could be successively transformed. Embryogenic calli of common bean were generated and transformed with the PvTRX1hRiA construction to down-regulate, by RNA interference, expression of the PvTRX1h gene. Plant hormone content was measured by mass spectrometry and gene expression was assessed by q-PCR. Detailed histological analysis was performed on selected transgenic embryogenic calli. It was determined that down-regulation of PvTRX1h gene was accompanied by altered concentrations of plant hormones in the calli. PvTRX1h regulated the expression of genes involved in auxin biosynthesis and embryogenic calli in which PvTRX1h was down-regulated were capable of differentiation into somatic embryos. Also, down-regulation of PvTRX1h showed increased transcript abundance of a gene coding for a second histone lysine methyltransferase, PvASHH2h. Accordingly, the PvTRX1h gene is involved in the synthesis of plant hormones in common bean callus. These results shed light on the crosstalk among histone methyltransferases and plant hormone signaling and on gene regulation during somatic embryo generation.

Ovariectomy-Induced Osteopenia Influences the Middle and Late Periods of Bone Healing in a Mouse Femoral Osteotomy Model.

PubMed

Pang, Jian; Ye, Meina; Gu, Xinfeng; Cao, Yuelong; Zheng, Yuxin; Guo, Hailing; Zhao, Yongfang; Zhan, Hongsheng; Shi, Yinyu

2015-08-01

It is known that bone healing is delayed in the presence of osteoporosis in humans. However, due to the complexities of the healing of osteoporotic fractures, animal models may be more appropriate for studying the effects of osteoporosis in more detail and for testing drugs on the fracture repair process. The purpose of this study was to investigate the influence of ovariectomy-induced osteopenia in bone healing in an open femoral osteotomy model, and to test the feasibility of this model for evaluating the healing process under osteopenic conditions. Ovariectomized (OVX) mouse models were employed to assess the effects of osteopenia on fracture healing, A mid-shaft femur osteotomy model was also established 3 weeks after ovariectomy as an osteopenic fracture group (OVX group). Femurs were then harvested at 2 weeks and 6 weeks after fracture for X-ray radiography, micro-computed tomography (micro-CT), histology, and biomechanical analysis. A sham-operated group (sham group) was used for comparison. The OVX mice had significantly lower bone volume density (BVF), volumetric bone mineral density (vBMD), and tissue mineral density (TMD) in the fracture calluses at 6 weeks (p<0.05), and similar trend was observed in 2 weeks. Additionally, larger calluses in OVX animals were observed via micro-CT and X-ray, but these did not result in better healing outcomes, as determined by biomechanical test at 6 weeks. Histological images of the healing fractures in the OVX mice found hastening of broken end resorption and delay of hard callus remodeling. The impaired biomechanical measurements in the OVX group (p<0.05) were consistent with micro-CT measurements and radiographic scoring, which also indicated delay in fracture healing of the OVX group. This study provided evidence that ovariectomy-induced osteopenia impair the middle and late bone healing process. These data also supported the validity of the mouse femoral osteotomy model in evaluating the process of bone healing under osteopenic conditions.

Ovariectomy-induced osteopenia influences the middle and late periods of bone healing in a mouse femoral osteotomy model.

PubMed

Pang, Jian; Ye, Meina; Cao, Yuelong; Zheng, Yuxin; Guo, Hailing; Zhao, Yongfang; Zhan, Hongsheng; Shi, Yinyu

2014-10-09

Objective It is known that bone healing was delayed in the presence of osteoporosis in humans. However, due to the complexities of the healing of osteoporotic fractures, animal models may be more appropriate to study the effects of osteoporosis in more details and to test drugs on the fracture repair process. The purpose of this study was to investigate the influence of ovariectomy-induced osteopenia in bone healing in an open femoral osteotomy model, and to test the feasibility of this model for evaluating the healing process under osteopenic conditions. Methods In assessing the effects of osteopenia on fracture healing, ovariectomized mouse models were employed. A mid-shaft femur osteotomy model was also established 3 weeks after ovariectomy as an osteopenic fracture group (OVX group). Femurs were then harvested at 2 weeks and 6 weeks after fracture for X-ray radiography, micro-computed tomography (micro-CT), histology and biomechanical analysis. A sham-operated group (Sham group) was used for comparison. Results The OVX mice had significantly lower BVF, vBMD and TMD in the fracture calluses at 6 weeks (P < 0.05), and similar trend was observed in 2 weeks. Additionally, larger calluses in OVX animals were observed via micro-CT and X-ray, but these did not result in better healing outcomes as determined by biomechanical test at 6 weeks. Histological images of the healing fractures in the OVX mice found forward of broken end resorption and delay of hard callus remodeling. The impaired biomechanical measurements in the OVX group (P < 0.05) were consistent with micro-CT measurements and radiographic scoring, which also indicated delay in fracture healing of the OVX group. Conclusions This study provided evidences that ovariectomy-induced osteopenia impair the middle and late bone healing process once more. These data also supported the validity of the mouse femoral osteotomy model in evaluating the process of bone healing under osteopenic conditions.

Genetic analysis of ectopic growth suppression during planar growth of integuments mediated by the Arabidopsis AGC protein kinase UNICORN.

PubMed

Enugutti, Balaji; Schneitz, Kay

2013-01-02

The coordination of growth within a tissue layer is of critical importance for tissue morphogenesis. For example, cells within the epidermis undergo stereotypic cell divisions that are oriented along the plane of the layer (planar growth), thereby propagating the layered epidermal structure. Little is known about the developmental control that regulates such planar growth in plants. Recent evidence suggested that the Arabidopsis AGC VIII protein kinase UNICORN (UCN) maintains planar growth by suppressing the formation of ectopic multicellular protrusions in several floral tissues including integuments. In the current model UCN controls this process during integument development by directly interacting with the ABERRANT TESTA SHAPE (ATS) protein, a member of the KANADI (KAN) family of transcription factors, thereby repressing its activity. Here we report on the further characterization of the UCN mechanism. Phenotypic analysis of flowers of ucn-1 plants impaired in floral homeotic gene activity revealed that any of the four floral whorls could produce organs carrying ucn-1 protrusions. The ectopic outgrowths of ucn integuments did not accumulate detectable signals of the auxin and cytokinin reporters DR5rev::GFP and ARR5::GUS, respectively. Furthermore, wild-type and ucn-1 seedlings showed similarly strong callus formation upon in vitro culture on callus-inducing medium. We also show that ovules of ucn-1 plants carrying the dominant ats allele sk21-D exhibited more pronounced protrusion formation. Finally ovules of ucn-1 ett-1 double mutants and ucn-1 ett-1 arf4-1 triple mutants displayed an additive phenotype. These data deepen the molecular insight into the UCN-mediated control of planar growth during integument development. The presented evidence indicates that UCN downstream signaling does not involve the control of auxin or cytokinin homeostasis. The results also reveal that UCN interacts with ATS independently of an ATS/ETT complex required for integument initiation and they further emphasize the necessity to balance UCN and ATS proteins during maintenance of planar growth in integuments.

Enhanced chondrogenesis and Wnt signaling in PTH-treated fractures.

PubMed

Kakar, Sanjeev; Einhorn, Thomas A; Vora, Siddharth; Miara, Lincoln J; Hon, Gregory; Wigner, Nathan A; Toben, Daniel; Jacobsen, Kimberly A; Al-Sebaei, Maisa O; Song, Michael; Trackman, Philip C; Morgan, Elise F; Gerstenfeld, Louis C; Barnes, George L

2007-12-01

Studies have shown that systemic PTH treatment enhanced the rate of bone repair in rodent models. However, the mechanisms through which PTH affects bone repair have not been elucidated. In these studies we show that PTH primarily enhanced the earliest stages of endochondral bone repair by increasing chondrocyte recruitment and rate of differentiation. In coordination with these cellular events, we observed an increased level of canonical Wnt-signaling in PTH-treated bones at multiple time-points across the time-course of fracture repair, supporting the conclusion that PTH responses are at least in part mediated through Wnt signaling. Since FDA approval of PTH [PTH(1-34); Forteo] as a treatment for osteoporosis, there has been interest in its use in other musculoskeletal conditions. Fracture repair is one area in which PTH may have a significant clinical impact. Multiple animal studies have shown that systemic PTH treatment of healing fractures increased both callus volume and return of mechanical competence in models of fracture healing. Whereas the potential for PTH has been established, the mechanism(s) by which PTH produces these effects remain elusive. Closed femoral fractures were generated in 8-wk-old male C57Bl/6 mice followed by daily systemic injections of either saline (control) or 30 microg/kg PTH(1-34) for 14 days after fracture. Bones were harvested at days 2, 3, 5, 7, 10, 14, 21, and 28 after fracture and analyzed at the tissue level by radiography and histomorphometry and at the molecular and biochemical levels level by RNase protection assay (RPA), real-time PCR, and Western blot analysis. Quantitative muCT analysis showed that PTH treatment induced a larger callus cross-sectional area, length, and total volume compared with controls. Molecular analysis of the expression of extracellular matrix genes associated with chondrogenesis and osteogenesis showed that PTH treated fractures displayed a 3-fold greater increase in chondrogenesis relative to osteogenesis over the course of the repair process. In addition, chondrocyte hypertrophy occurred earlier in the PTH-treated callus tissues. Analysis of the expression of potential mediators of PTH actions showed that PTH treatment significantly induced the expression of Wnts 4, 5a, 5b, and 10b and increased levels of unphosphorylated, nuclear localized beta-catenin protein, a central feature of canonical Wnt signaling. These results showed that the PTH-mediated enhancement of fracture repair is primarily associated with an amplification of chondrocyte recruitment and maturation in the early fracture callus. Associated with these cellular effects, we observed an increase in canonical Wnt signaling supporting the conclusion that PTH effects on bone repair are mediated at least in part through the activation of Wnt-signaling pathways.

Genetic fidelity assessment of in vitro-regenerated plants of Albizia julibrissin using SCoT and IRAP fingerprinting

Treesearch

Mohammad-Shafie Rahmani; Paula M. Pijut; Naghi Shabanian; Mona Nasri

2015-01-01

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in...

Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens

USDA-ARS?s Scientific Manuscript database

Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait loc...

Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.)

Treesearch

G. Vengadesan; Paula M. Pijut

2009-01-01

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) after 4 weeks of...

Somaclonal variation in hybrid poplars for resistance to Septoria leaf spot

Treesearch

M.E. Ostry; D. D. Skilling

1987-01-01

Tissue culture techniques have been used to obtain hybrid poplars with putative resistance to leaf spot caused by Septoria musiva from clones previously susceptible to the disease. Stem internode explants were used to obtain proliferating callus cultures. Adventitious bud formation and shoot proliferation were then induced. Elongated shoots were excised and rooted in a...

Glycoprotein of the wall of sycamore tissue-culture cells.

PubMed

Heath, M F; Northcote, D H

1971-12-01

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.

Endophytic Penicillium citrinum Thom. from Scoparia dulcis Linn.

PubMed

Mathew, Annie J; Jayachandran, K; Mathew, Jyothis

2010-10-01

Scoparia dulcis of Scrophulariaceae is an annual herb distributed through out the tropics. Penicillium citrinum was obtained from apparently healthy roots, stem, leaves and fruits of this plant. Callus and multiple shoots produced during micropropagation from various explants were also symptomless but showed occurrence of Penicillium citrinum when cultured in Murashige & Skoog liquid medium for the production of secondary metabolites.

Enhanced pest resistance and increased phenolic production in maize callus transgenically expressing a maize chalcone isomerase -3 like gene

USDA-ARS?s Scientific Manuscript database

Significant losses in maize production are due to damage by insects and ear rot fungi. A gene designated as chalcone-isomerase-like, located in a quantitative trait locus for resistance to Fusarium ear rot fungi, was cloned from a Fusarium ear rot resistant inbred and transgenically expressed in mai...