Callus Structure Research Articles

Silver Nanoparticles (AgNPs) Act as Nanoelicitors in Melissa officinalis to Enhance the Production of Some Important Phenolic Compounds and Essential Oils

ABSTRACTNanoparticles (NPs) are well‐known biostimulants in plant biotechnology, utilised to enhance the physical properties of plants and exhibit positive effects on them. The important key role is the most suitable type, effective dose and size of NP to be used in plant tissue culture systems. In this study, various concentrations of silver nanoparticles (AgNPs; 0, 25, 50, 75 and 100 μg L−1) were tested as elicitors in callus culture with the aim of enhancing secondary metabolite production in lemon balm (Melissa officinalis L.). According to the results obtained, callus formation rates have shown an increase in all applications compared to the control group. The highest callus formation, weight and diameter were observed in 50 μg L−1 application. In this application, the callus structure was compact and its colour was green. However, the aromatic compounds, neral and geranial increased significantly in 25 μg L−1 application. The maximum increase in phenolic compounds such as caffeic acid, chlorogenic acid, proto‐catechic acid, hesperidin and p‐coumaric acid was observed in the 75 μg L−1 AgNP and the highest increase in rosmarinic acid compound was determined in the 50 μg L−1 application. The study found that AgNP applications are an effective method for increasing the production of secondary metabolites in medicinal and aromatic plants, such as lemon balm, in vitro.

Callus Regeneration of Gynura procumbens (Lour.) Merr. In Vitro

Gynura procumbens (Lour.) Merr. is one of the species of Asteraceae which is potential as medicinal. Propagation of the species could be conducted by vegetative, so the plant genetic variability is narrow. Genetic variability could be increased by somaclonal variation through callus culture. There is no report on in vitro regeneration from callus culture, although this method could assist further genetic improvement of plants. In this experiment, different concentrations of 2,4- D (0.1 ; 0.3 ; 0.5 mg L -1 ) singly or combination with BA (0.1 ; 0.3 and 0.5 mg L -1 ) were evaluated for callus induction and several concentrations of BA (0 ; 0.1; 0.5 and 1.0 mg -1 ) combination with kinetin (0.1 and 0.3 mg-1) were observed for ability of callus formed shoots. The results showed that the best media for callus induction was 0.5 mg L -1 2,4-D + 0.5 mg L -1 BA. This treatment produced friable callus structure, no roots and yellowish white. Callus regeneration was obtained on the combination of 0.1 mg L -1 BA. + 0.1 mg L -1 kinetin and 0.1 mg L -1 BA + 0.5 mg L -1 kinetin but the percentage was still low. Keywords: G procumbens {Lour.} Merr, induction, regeneration, calli, in vitro

Embryogenic Callus Induction of Todolo Toraja Coffee Leaf Cells (Coffea arabica Var. Typica) with the Addition of 2, 4-DichlorophenoxyaceticAcid (2, 4-D) and Furfurylaminopurine (Kinetin) in Vitro

Somatic embryogenesis is a way to produce large numbers of seeds according to the parent, so it is very profitable in the effort to conserve Arabica coffee, which has reduced number of trees and is not productive. This study uses Todolo arabica coffee (Coffea arabica Var. Typica) which is the oldest commercial coffee found in Toraja, South Sulawesi and is threatened with extinction. This study aims to obtain the best composition in administering zpt 2,4-D and kinetin in forming embryogenic callus from explants of arabica coffee leaves. This research was conducted at the Tissue Culture Laboratory of the Faculty of Mathematics and Natural Sciences, Hasanuddin University. The experimental range used was a completely randomized design with 2 factorials, namely the concentration of 2,4-D 1 ppm, 2 ppm, and 3 ppm and kinetin concentration of 0.1 ppm, 0.5 ppm and 1.0 ppm so that there were 9 treatments with 3 replications. The parameters observed were callus formation time, callus structure, and callus color. The results showed that the fastest time for callus appearance was 17.00 HSK with 1 ppm 2,4-D and 0.5 ppm kinetin. The callus texture formed is a crumb-textured callus with several color variations that are formed.

Cyto-histological aspects of haploid androgenesis when obtaining haploids/doubled haploids in rice (Oryza Sativa l.) anther culture in vitro

The aim of the study was to study the development of rice microspores in anther culture in vitro, study the structure of androclinic callus to identify cyto-embryological features of the formation of morphogenic structures in anther culture, obtain doubled rice haploids (Oryza sativa L.) and accelerate the development of valuable breeding material with desired properties. Within the framework of this study, the results of a cyto-histological study of rice haploid androgenesis in vitro were obtained, which indicate that it is induced under the influence of phytohormones from rice anther microspores at the mononuclear or early binuclear stage of development. The abnormal development of microspores on nutrient media with phytohormone 2,4-D was traced, in which nuclei, having lost their characteristic functions, acquired the ability to unlimited division and growth with the formation of microcallus. The morphological structure of calli was assessed. The main morphotypes of callus tissues and the pathways of morphogenesis, leading to the formation of androgenic structures, up to full-fledged regenerant plants, were identified. Homozygous androgenic lines based on F1 and BC1 - rice generations obtained in the course of hybridization and backcrossing between Chinese samples carrying blast resistance genes and Russian accessiond were rapidly developed.

The First Experience of Dynamic Intramedullary Osteosynthesis of Diaphyseal Fractures of the Tibia Using Implants Based on Magnesium Alloy

Summary. The efficiency of using the dynamic blocking intramedullary osteosynthesis (BIOS) technique for tibial fractures with the use of biodegradable implants based on the MS-10 magnesium alloy was determined.
 Objective: to approve the technique and to study the effectiveness of dynamic intramedullary osteosynthesis of diaphyseal fractures of the tibia using locking screws made of the biodegradable magnesium alloy MS-10.
 Materials and Methods. The study was carried out in the Department of Traumatology of the Municipal Non-Profit Enterprise “City Hospital of Emergency and Ambulance” of Zaporizhzhia City Council in Zaporizhzhia on 15 patients with diaphyseal fractures of the tibia, who underwent BIOS. Cannulated intramedullary titanium rods were used as a fixator. In the proximal section, a screw made of titanium alloy was introduced into a dynamic hole; a screw made of a biodegradable magnesium alloy MC-10 was inserted into a static hole. In the postoperative period, the patients underwent a course of complex rehabilitation measures. X-ray control was performed on day 1, at week 4, week 8, and week 16 after the surgery.
 Results. After the start of the dosed load on the injured limb, a fracture of the locking screw made of MC-10 alloy was radiographically recorded in all patients. The dynamic screw moves in the dynamic hole and the fracture is dynamized. The formation of callus in the fracture zone was unremarkable and took an average physiological time. The quality and structure of callus did not show any pathological features. Four months after the surgery, all patients were socially adapted and did not use additional support when moving.
 Conclusions. Based on the data obtained, it is possible to draw a conclusion about the possibility and advisability of using biodegradable implants made of magnesium alloy MS-10 in a dynamic BIOS of long bones of the human skeleton.

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS + TDZ, and ½ MS + IAA + TDZ + PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS + IAA + TDZ + PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS + IAA + TDZ and ½ MS + IAA + TDZ + PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.

A C-Met chemical inhibitor promotes fracture healing through interacting with osteogenic differentiation via the mTORC1 pathway

Currently, HGF/C-Met signaling inhibitors are being investigated to determine if they are useful for enhancing progenitor cell differentiation into osteoblasts, and one of them, BMS-777607, has been utilized to treat osteoporosis and bone loss in several types of diseases. However, whether BMS-777607 could be a potential treatment during fracture healing remains elusive. Here, we examined the therapeutic effects of BMS-777607 on bone fracture healing in a mouse model. In vivo radiological analysis showed that fractures treated with BMS-777607 exhibited accelerated osteotylus formation during the early stage of bone healing. Thereafter, the Safranin O staining evaluation indicated that the structure of the external callus in the Treatment group was larger than that in the Vehicle group at week 2. Furthermore, cellular proliferation of MC3T3-E1 was not significantly affected by low concentrations of BMS-777607. In addition, stimulation of osteoblast differentiation and mineralization was a result of BMS-777607 inducing the expression of Runx2 and Col1, and this osteogenic ability, at least in part, was mediated through the mammalian target of rapamycin complex 1 (mTORC1) signaling in vitro. Conclusively, BMS-777607 has been identified as a therapeutic agent to improve bone formation during fracture healing, and its osteogenic effects on osteoblast differentiation were mediated via the mTORC1 signaling pathway.

The role of nanosilver in improving frequency of single cell formation of Limonium sinuatum (L.) Mill

In this research, nanosilver with different concentrations and treatment time was used to sterilize infection agents and induce initial explants which were used as materials for cell suspension culture of Limonium sinuatum. The result showed that leaf explants sterilized with 0.2 g/L nanosilver for 20 minutes had highest effect (live rate 73.33%) comparing to HgCl2and Ca(ClO)2(56.66% and 64.44%, respectively). In addition, the leaf explants which were treated with nanosilver and cultured in ½ MS medium supplemented with 20 g/L sucrose, 1 mg/L picloram and 2.5 g/L gelrite also induced calli (friable calli, milk white color, embryogenic callus structure). Moreover, cell suspension formation process from these calli was also observed highest on the 20thday (49,088 cells/mL) in liquid shaking culture condition comparing to control treatment (19,361 cells/mL) when they were cultured on similar medium combined with 1.2 mg/Lnanosilver. These cells had the best growth, development and regeneration from the 16thday to the 20thday. The ability of shoot and callus regeneration was highest (67.77%) in ½ MS medium which was supplemented with 20 g/L sucrose, 1 mg/L zeatin, 2.5 g/L gelrite and 1.6 mg/L nanosilver compared with control treatment having no nanosilver (40.00%). This research showed that in micropropagation of Limonium sinuatum, nanosilver was proved to be an effective agent for sterilization, explant induction, cell suspension origination, and callus and shoot regeneration. Beside that, nanosilver had negative impact on the development of explants when it was used with high concentration for extended period.

Experimental examination of pollinator-mediated selection in a sexually deceptive orchid.

Selection exerted by pollinators on flowers is predicted to occur along two distinct axes. While pollinator attraction to flowers is governed by pollinator preferences, pollen transfer efficiency is mediated by the mechanical fit of pollinators to flower morphology. Although pollinator attraction in sexually deceptive orchids is typically underpinned by floral odour, morphological traits are expected to play a vital role in mechanical fit during floral contact with pollinators. Here we utilize a comprehensive and novel procedure to test for pollinator-mediated selection through mechanical fit with the flower labellum in the orchid Chiloglottis trapeziformis. This approach combines detailed pollinator observations related to plant reproductive fitness with complementary experimental manipulation and phenotypic selection analysis. Experiments with virgin flowers revealed that pollen removal occurs only during vigorous pseudocopulation. This behaviour involves male wasps that grasp the insectiform callus structure on the labellum while probing the labellum tip in a forward orientation. Both orientation and duration of pseudocopulation were significant predictors of pollen removal, confirming a direct relationship between pollinator behaviour and plant fitness. Controlled floral manipulation that either shortened or elongated the distance between the callus and the labellum tip detected no change in pollinator attraction. The duration of pseudocopulation, however, was significantly reduced on flowers with shortened or elongated callus-tip distances, consistent with stabilizing selection. Phenotypic selection analysis confirmed this prediction in natural populations by uncovering evidence for stabilizing selection on the distance between the callus and the labellum tip. Our experimental manipulations and selection analysis in natural populations thus demonstrate stabilizing selection on the distance from the callus to the labellum tip, and illustrate the utility of employing multiple approaches to confirm selection exerted by pollinators on floral form.

Somatic embryogenesis of East Kalimantan local upland rice varieties

Somatic embryogenesis is the formation, growth and development of embryos from somatic cells. Somatic embryo induction is one of the in vitro plant propagation techniques that is very important for plant developmental purposes. Four local upland rice varieties of East Kalimantan, Mayas Pancing, Gedagai, Siam and Serai, were used in this study. A total of 200 explants (mature rice grains) for each varieties were inoculated on MS solid medium supplemented with 1 mg L-1 2,4 Dichlorophenoxy acetic acid (2,4-D) and 0.5 mg L-1 6-Benzylaminopurine (BAP). The results showed that response of each variety differed to embryosomatic induction, indicated by callus induction rate and callus quality, in terms of callus color and structure. The fastest callus formation was sobserved in Gedagai variety (8 days) while Mayas Pancing (13 days) was the latest one. The rate of callus induction varied from 60 to 98.5 %, and Serai variety has the highest callus induction rate. The highest friable callus structure was found in Siam variety (89.1%) and the lowest was in Gedagai (62.5%). Callus color was dominated by the yellowish-white (transparent) on all varieties tested. Most of the callus was potential as embryogenic callus characterized from the nodular and globular of friable callus structure and its yellowish-white color.

Using 3D finite element models verified the importance of callus material and microstructure in biomechanics restoration during bone defect repair

Background: There is lack of further observations on the microstructure and material property of callus during bone defect healing and the relationships between callus properties and the mechanical strength. Methods: Femur bone defect model was created in rabbits and harvested CT data to reconstruct finite element models at 1 and 2 months. Three types of assumed finite element models were compared to study the callus properties, which assumed the material elastic property as heterogeneous (R-model), homogenous (H-model) or did not change from 1 to 2 months (U-model). Results: The apparent elastic moduli increased at 2 months (from 355.58 ± 132.67 to 1139.30 ± 967.43 MPa) in R-models. But there was no significant difference in apparent elastic moduli between R-models (355.58 ± 132.67 and 1139.30 ± 967.43 MPa) and H-models (344.79 ± 138.73 and 1001.52 ± 692.12 MPa) in 1 and 2 months. A significant difference of apparent elastic moduli was found between the R-model (1139.30 ± 967.43 MPa) and U-model group (207.15 ± 64.60 MPa) in 2 months. Conclusions: This study showed that the callus structure stability remodeled overtime to achieve a more effective structure, while the material quality of callus tissue is a very important factor for callus strength. At the meantime, this study showed an evidence that the material heterogeneity maybe not as important as it is in bone fracture model.

Numerical evaluation of the backward propagating acoustic field in healing long bones.

The propagation of ultrasound in healing long bones induces complex scattering phenomena due to the interaction of an ultrasonic wave with the composite nature of callus and osseous tissues. This work presents numerical simulations of ultrasonic propagation in healing long bones using the boundary element method aiming to provide insight into the complex scattering mechanisms and better comprehend the state of bone regeneration. Numerical models of healing long bones are established based on scanning acoustic microscopy images from successive postoperative weeks considering the effect of the nonhomogeneous callus structure. More specifically, the scattering amplitude and the acoustic pressure variation are calculated in the backward direction to investigate their potential to serve as quantitative and qualitative indicators for the monitoring of the bone healing process. The role of the excitation frequency is also examined considering frequencies in the range 0.2-1 MHz. The results indicate that the scattering amplitude decreases at later stages of healing compared to earlier stages of healing. Also, the acoustic pressure could provide supplementary qualitative information on the interaction of the scattered energy with bone and callus.

Proliferation of embryogenic callus of Satoimo taro (Colocasia esculenta var. antiquorum) in culture media with various levels of sucrose and gelling agent

Abstract. Fitriani H, Aryaningrum PD, Hartati NS. 2016. Proliferation of embryogenic callus of Satoimo taro (Colocasia esculenta var. antiquorum) in culture media with various level of sucrose and gelling agent. Nusantara Bioscience 8: 316-320. Satoimo taro (Colocasia esculenta var. antiquorum) is one of the promising varieties of taro which have potentially high value to cultivate. Tissue culture or in-vitro culture considers an economically useful technique to mass propagates this variety of taro. The research was conducted to figuring out the optimum media for proliferation success of embryogenic callus of Satoimo. The explants used embryogenic callus of 12-months-old storage materials obtained from the previous study. Proliferation media include a basal half-strength MS salt (Murashige and Skoog) enriched with of 0.1 mg/L TDZ, 0.05 mg/L 2.4 D, and 100 mg/L L-glutamine. Adding to the basal media, we set the treatments by combining two factors i.e. Sucrose with three levels of concentration i.e. 3, 4, and 5% which cross-tabulated with three gelling agents, i.e., Agarose, Phytagel, and Gerlite. The research parameters included the percentage of callus diameter growth, the color and callus discoloration, and the structure of callus. The data recorded periodically every 7 d in one month. Data were analyzed using analysis of variance and followed by Duncan Multiple Range Test (DMRT) to determine the significant difference (p<0.05) among the treatments. The result revealed that the callus diameter growth has the greatest percentage at medium with a combination of Phytagel and sucrose 4%. The Micro-agar enhanced the presence of callus with yellow color. Phytagel combined with a high percentage of sucrose (5%) indicated browning properties of callus. Gelrite causes the callus with discoloration appearance (white).

The osteogenic potential of human bone callus.

Bone callus, generated during fracture healing, is commonly discarded during surgical procedures. The aim of this study was to investigate the osteogenic potential of bone callus and its possible use as autograft material for patients needing bone grafts. Histology, immunohistochemistry, micro-computed tomography, and biomechanics were performed to examine osteogenic cells, osteoinductive factors, and the osteoconductive structure of bone callus. Alkaline phosphatase-positive osteoblasts, osteoinductive factors (including BMP2, FGF2, TGFB1, and IGF1), and a porous structure were found in bone callus. Early-stage callus (within 3 months after fracture) presented significantly improved osteogenic properties compared to medium- (3–9 months) and late-stage (longer than 9 months) callus. The results revealed that bone callus induced new bone formation in a nude mouse model. Early-stage callus showed better performance to medium- and late-stage callus in the induction of new bone formation at both 8 and 12 weeks. These findings indicated that bone callus, especially early-stage callus, possesses osteogenic potential and can potentially serve as an alternative source of material for bone grafts.

Effect of combined treatment with zoledronic acid and parathyroid hormone on mouse bone callus structure and composition

In recent years, great interest in combined treatment of parathyroid hormone (PTH) with anti-resorptive therapy has emerged. PTH has been suggested to aid bridging of atrophic fractures and improve strength in closed fracture models. Bisphosphonate treatments typically result in a larger woven bone callus that is slower to remodel. The combination of both drugs has been demonstrated to be effective for the treatment of osteoporotic bone loss in many preclinical studies. However, the effect of combined treatment on fracture repair is still largely unexplored. In this study, we aimed to compare these drugs as single-agent and in combination in a murine closed fracture model. We wanted to assess potential differences in material properties, morphometry and in the development of the lacuno-canalicular network. A total of 40 female, 11-week-old wild type mice underwent a closed fracture on the midshaft of the tibia and were assigned to four groups (n=8–10 per group). Beginning on post-operative day 8, animals received different subcutaneous injections. Group 1 received a single injection of saline solution and Group 2 of zoledronic acid (ZA). Group 3 received daily dosing of PTH. Group 4 received a dual treatment, starting with a single dose of ZA followed by daily injection of PTH. Three weeks after fracture, all animals were euthanized and tibiae were assessed using micro-computed tomography (micro-CT), high-resolution micro-CT (HR micro-CT), Raman spectroscopy, quantitative histomorphometry, and deconvolution microscopy (DV microscopy). Combined treatment showed a significant increase of 41% in bone volume fraction and a significant decrease of 61% in the standard deviation of the trabecular spacing compared to vehicle, both known to be strong predictors of callus strength. An analysis via HR micro-CT showed similar results on all groups for lacunar numerical density, whereas mean lacuna volume was found to be higher compared to vehicle in treated groups, but only PTH mono-treatment showed a significant increase compared to vehicle (+45%). Raman spectroscopy did not reveal detectable changes in material properties of the bone calluses. Sclerostin staining, tartrate resistant acid phosphatase (TRAP) staining and canalicular analysis with DV microscopy on a subset of samples did not display distinctive difference in any of the treatments.We therefore consider PTH+ZA treatment beneficial for bone healing. No clear negative effect on bone quality was detected during this study.

4. The Low-Intensity Pulsed Ultrasound (LIPUS) Mechanism and the Effect of Teriparatide on Fracture Healing.

Low-Intensity Pulsed Ultrasound (LIPUS) provided a mechanical stimulus, and was thought to promote fracture healing by signal transduction through integrin, a cytoskeletal protein. Meanwhile, teriparatide, a drug for osteoporosis treatment, showed efficacy in promoting bone metabolism. This drug also appeared to prevent fractures in patients with serious osteoporosis by improving bone mineral density and bone quality, which in turn resulted from promoting action for bone metabolism. Further, clinical trials and fundamental research reported that teriparatide demonstrated the effect of promoting fracture healing. Mechanical stimulus by LIPUS had a topical effect on fractures; on the other hand, teriparatide (peptide hormone) had both topical and systemic effects. Both LIPUS and teriparatide had the effect of fracture healing, but it was supposed that the characteristics of each effect were different because of the different mechanism of action. Moreover, the combination therapy of LIPUS and teriparatide was expected to produce synergies. We used elderly rats as models for the femoral fracture to examine the effects of LIPUS and teriparatide on promoting fracture healing for treatment delay by aging. We observed the fracture healing process in 40-week-old rats as an elderly model using simple radiographs, and recognized a delay in fracture healing compared with that of 8-week-old rats. As discussed in histomorphology, it was demonstrated that the period of endochondral ossification, from chondrogenesis to teleost cross-linked callus, was prolonged and the fracture healing process was delayed by aging. Next, we treated the elderly fracture models with LIPUS for 20 minutes a day from the first day after the fracture, and compared them with non-treated models. The bone unions of the treated models were observed earlier than those of non-treated models in the simple radiographs. LIPUS shortened the period of endochondral ossification. Further, we gave the elderly fracture models teriparatide subcutaneously 5 μg/kg three times a week from the first day after the fracture. Bone unions of the treated models were observed earlier than those of non-treated models in simple radiographs as well. In micro CT analysis, it was demonstrated that lamellar bone transforming and bone remodeling of the trabecular structure of external callus were especially accelerated. The results of these trials showed that both LIPUS and teriparatide demonstrated the effect of promoting fracture healing, and each had unique characteristics.

RolB gene-induced production of isoflavonoids in transformed Maackia amurensis cells.

Maackia amurensis Rupr. et Maxim is a valuable leguminous tree grown in the Russian Far East, in China, and in Korea. Polyphenols from the heartwood of this species (primarily stilbenes and isoflavonoids) possess strong hepatoprotective activity. Callus culture of M. amurensis produced isoflavonoids and their derivatives. In pharmacological experiments, the callus complex was at least as effective, as the plant complex. To increase the yield of isoflavonoids, calli were transformed with the rolB gene of Agrobacterium rhizogenes. Neomycin phosphotransferase (nptII) gene was used for transgenic cell selection. Three rolB transgenic callus lines with different levels of the rolB gene expression were established. Insertion of the rolB gene caused alterations in callus structure, growth, and isoflavonoid production, and stronger alterations were observed with higher expression levels. MB1, MB2, and MB4 cultures accumulated 1.4, 1.5, and 2.1% of dry weight (DW) isoflavonoids, respectively. In contrast, the empty vector-transformed MV culture accumulated 1.22% DW. Isoflavonoid productivity of the obtained MB1, MB2, and MB4 cultures was equal to 117, 112, and 199mg/L of medium, respectively, comparing to 106mg/L for the MV culture. High level of expression of the rolB gene in MB4 culture led to a 2-fold increase in the isoflavonoid content and productivity and reliably increased dry biomass accumulation. Lower expression levels of the rolB gene in MB1 and MB2 calli did not significantly enhance biomass accumulation and isoflavonoid content, although the rolB gene activated isoflavonoid biosynthesis during the early growth stages and caused the increased content of several distinct compounds.

Embryogenic Callus Induction of Pencil Orchid (Papilionanthe hookeriana Rchb.f.) Through in Vitro Culture

Papilionthe hookeriana Rchb.f. (Orchidaceae), popularly known as ‘Anggrek Pinsil’ in Indonesia, is a perennial ephiphyte orchid, found only at Dendam Tak Sudah Lake in Bengkulu. The aims of this research were to find the best sterilization technique of P. hookeriana explant and to induce maximum formation of embryogenic calli. Rapid multiplication of this orchid was achieved through culture of shoot tips and young leaf segments of mature plants by in vitro cultured on Murashige and Skoog (MS) basal medium enriched with 50 g L -1 banana pulp. The experiment used Completely Randomized Design (CRD) with three replications. In the first stage, the explants were sterilized using three compositions of sterilant. In the second stage, the explants were planted on the MS basal medium with addition of five levels of 2,4-Diclhorophenoxy Acetis Acid concentrations, namely 0, 0.25, 0.50, 0.75, and 1.00 mg L -1 . The result showed that the best sterilization method to reduce explant contaminant was method 3, in which the explant was washed with detergent, rinsed with flowing water, soaked in 0.1% (v/v) HgCl 2 solution for 30 minutes, soaked again in the 10% (v/v) Natrium hypocloride solution for 20 minutes, rinsed three times with sterile water before planted and then soaked in sterile water + 10% (v/v) betadhine before planted on treated medium. This method was able to reduce contamination levels up to 70% from explants cultured for 5 months on MS medium. MS medium added with 1.00 mg L -1 2,4-D produced the highest number of embryogenic calli, and the biggest callus diameter ( 3.5 cm ), characterized by transparent green color and friable callus structure

A new large-flowered odontoglossum (orchidaceae: oncidiinae) from the Cusco region in Peru

Plants of a colorful Odontoglossum were recently found by separate parties in the Cusco region of central Peru. The species was incorrectly identified as Odontoglossum epidendroides, independently by both parties. Although similar in appearance, a closer examination of the flower reveals that it represents a new species, which is scientifically described here and compared with similar species. The new taxon is distinguished from the similar O. cruentum and O. juninense by morphological features of the column, such as the lateral curvature profile, with larger and more rectangular wings, and in combination with the well developed falcate callus structure on the lip. Our new species is also distinguished from the rather similar O. epidendroides by having flowers with a shorter column with shorter wings.

Evaluation of composition and mineral structure of callus tissue in rat femoral fracture

Callus formation is a critical step for successful fracture healing. Little is known about the molecular composition and mineral structure of the newly formed tissue in the callus. The aim was to evaluate the feasibility of small angle x-ray scattering (SAXS) to assess mineral structure of callus and cortical bone and if it could provide complementary information with the compositional analyses from Fourier transform infrared (FTIR) microspectroscopy. Femurs of 12 male Sprague-Dawley rats at 9 weeks of age were fractured and fixed with an intramedullary 1.1 mm K-wire. Fractures were treated with the combinations of bone morphogenetic protein-7 and/or zoledronate. Rats were sacrificed after 6 weeks and both femurs were prepared for FTIR and SAXS analysis. Significant differences were found in the molecular composition and mineral structure between the fracture callus, fracture cortex, and control cortex. The degree of mineralization, collagen maturity, and degree of orientation of the mineral plates were lower in the callus tissue than in the cortices. The results indicate the feasibility of SAXS in the investigation of mineral structure of bone fracture callus and provide complementary information with the composition analyzed with FTIR. Moreover, this study contributes to the limited FTIR and SAXS data in the field.