Ploidy Status, Nuclear DNA Content and Start Codon Targeted (SCoT) Genetic Homogeneity Assessment in Digitalis purpurea L., Regenerated In Vitro.

Digitalis purpurea L. is a therapeutically important plant that synthesizes important cardiotonics such as digitoxin and digoxin. The present work reports a detailed and efficient propagation protocol for D. purpurea by optimizing various PGR concentrations in Murashige and Skoog (MS) medium. The genetic homogeneity of in vitro regenerants was assessed by the flow cytometric method (FCM) and Start Codon Targeted (SCoT) marker technique. Firstly, the seeds inoculated in full MS medium added with 0.5 mg/L GA3 produced seedlings. Different parts such as hypocotyl, nodes, leaves and apical shoots were used as explants. The compact calli were obtained on BAP alone or in combinations with 2, 4-D/NAA. The hypocotyl-derived callus induced somatic embryos which proliferated and germinated best in 0.75 mg/L BAP-fortified MS medium. Scanning electron microscopic (SEM) images confirmed the presence of various developmental stages of somatic embryos. Shoot regeneration was obtained in which BAP at 1.0 mg/L and 2.0 mg/L BAP + 0.5 mg/L 2,4-D proved to be the best treatments of PGRs in inducing direct and indirect shoot buds. The regenerated shoots showed the highest rooting percentage (87.5%) with 24.7 ± 1.9 numbers of roots/shoot in 1.0 mg/L IBA augmented medium. The rooted plantlets were acclimatized in a greenhouse at a survival rate of 85-90%. The genome size and the 2C nuclear DNA content of field-grown, somatic embryo-regenerated and organogenic-derived plants were estimated and noted to be 3.1, 3.2 and 3.0 picogram (pg), respectively; there is no alteration in ploidy status and the DNA content, validating genetic uniformity. Six SCoT primers unveiled 94.3%-95.13% monomorphic bands across all the plant samples analyzed, further indicating genetic stability among in vitro clones and mother plants. This study describes for the first time successful induction of somatic embryos from hypocotyl callus; and flow cytometry and SCoT marker confirmed the genetic homogeneity of regenerated plants.

Direct somatic embryogenesis and flow cytometric assessment of ploidy stability in regenerants of Caladium × hortulanum 'Fancy'.

Caladium × hortulanum 'Fancy' is an important ornamental plant grown in pots and landscapes and known for its colorful leaves often used for interior decorations. In this work, we present a method of in vitro regeneration from three explants source through direct somatic embryogenesis (DSE) wherein the regenerated plants were screened for ploidy changes through flow cytometry analysis. Tuber, leaf and petiole explants were cultured on MS basal medium supplemented with 1-napthalene acetic acid (NAA), 6-benzyl amino purine (BAP) and N-phenyl-N'-1, 2,3-thiadiazol-5-ylurea (TDZ) concentrations. Tuber explants induced highest direct somatic embryos on NAA (1 mg L- 1) + BAP (0.5 mg L- 1) with 55.6 mean number of embryos per explant while as leaf and petiole explants amended with 1 mg L- 1 TDZ developed 18.7 and 12.27 mean number of embryos per explant respectively. The highest embryo conversion frequency was achieved on BAP (2 mg L- 1) + NAA (0.2 mg L- 1) with 44.2, 18.7 and 7.5 mean number of plantlets produced per tuber, leaf and petiole explant respectively after 4 weeks of culture. Plantlets were later rooted and maximum number of roots (6.33) per shoot was achieved on 2 mg L- 1 indolebutyric acid amended medium. Description of the process of DSE is presented through the histological and SEM evidences. The 2C DNA content of field grown plants and the DSE regenerants evaluated under flow cytometric analysis were 8.06 pg and 8.28 pg respectively showing no ploidy changes. Hence, a successful protocol of inducing direct somatic embryos from three explant types with efficient embryo conversion frequency was obtained with regenerants showing similar DNA ploidy as that of their parent plants.

Mass propagation through direct and indirect organogenesis in three species of genus Zephyranthes and ploidy assessment of regenerants through flow cytometry.

Genus Zephyranthes consists of economically important plant species due to their high ornamental value and presence of valuable bioactive compounds. However, this genus propagates by asexual division only which gives slow propagation rate. Plant tissue culture has the potential to provide efficient techniques for rapid multiplication and genetic improvement of the genus. In this work, a dual in vitro regeneration system through callus mediated shoot regeneration and direct shoot regeneration in species Zephyranthes candida, Zephyranthes grandiflora and Zephyranthes citrina was investigated. Bulb, leaf and root explants were cultured on Murashige and Skoog (MS) medium amended with different plant growth regulators (PGR's) viz. 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), N-phenyl-N'-1,2,3 -thiadiazol-5-ylurea (TDZ), 6-Furfuryl- aminopurine (KIN) alone or in combinations for callus induction and regeneration. Only bulb explants showed callus induction and regeneration response on different PGR combinations with a varied response in callus induction percentage, callus color and callus texture. Creamish compact callus (CC) was induced on 2 mg L[Formula: see text] 2,4-D, brown friable callus (BF) on 2 mg L[Formula: see text] NAA + 1 mg L[Formula: see text] BAP and green friable callus (GF) callus on 1 mg L[Formula: see text] KIN + 3 mg L[Formula: see text] NAA. The maximum shoot multiplication from different callus types (indirect organogenesis) was achieved on 2 mg L[Formula: see text] BAP alone without combinations. Bulb explants of Z. grandiflora induced maximum callus induction percentage (86.4%) and shoot regeneration percentage (83.5%) with the maximum 08 shoots per 150 mg callus mass. The induction and regeneration response was followed in the order of Z. grandiflora > Z. candida > Z. citrina. Similarly, maximum direct organogenesis from bulb explants was obtained in Z. grandiflora (93.3%) followed by Z. candida (91.5%) and Z. citrina (90.4%) on 3 mg L[Formula: see text] TDZ amended MS media. Adventitious root induction was achieved on 2 mg L[Formula: see text] IBA with a maximum of 8 roots per shoot. The in vitro raised plantlets were successfully acclimatized in the field with 85% survival efficiency. The genome size (2C DNA content) of the field-grown plants and in vitro regenerated plants, evaluated through flow cytometry technique, were similar and showed no ploidy changes. An efficient mass propagation protocol was established for obtaining plants with unaltered genome size in the three species of Zephyranthes.

A Rabbit Femoral Condyle Defect Model for Assessment of Osteochondral Tissue Regeneration.

Osteochondral tissue repair represents a common clinical need, with multiple approaches in tissue engineering and regenerative medicine being investigated for the repair of defects of articular cartilage and subchondral bone. A full thickness rabbit femoral condyle defect is a clinically relevant model of an articulating and load bearing joint surface for the investigation of osteochondral tissue repair by various cell-, biomolecule-, and biomaterial-based implants. In this protocol, we describe the methodology and 1.5- to 2-h surgical procedure for the generation of a reproducible, full thickness defect for construct implantation in the rabbit medial femoral condyle. Furthermore, we describe a step-by-step procedure for osteochondral tissue collection and the assessment of tissue formation using standardized histological, radiological, mechanical, and biochemical analytical techniques. This protocol illustrates the critical steps for reproducibility and minimally invasive surgery as well as applications to evaluate the efficacy of cartilage and bone tissue engineering implants, with emphasis on the usage of histological and radiological measures of tissue growth. Impact statement Although multiple surgical techniques have been developed for the treatment of osteochondral defects, repairing the tissues to their original state remains an unmet need. Such limitations have thus prompted the development of various constructs for osteochondral tissue regeneration. An in vivo model that is both clinically relevant and economically practical is necessary to evaluate the efficacy of different tissue engineered constructs. In this article, we present a full thickness rabbit femoral condyle defect model and describe the analytical techniques to assess the regeneration of osteochondral tissue.

Assessment of cartilage regeneration on 3D collagen-polycaprolactone scaffolds: Evaluation of growth media in static and in perfusion bioreactor dynamic culture.

Efforts on bioengineering are directed towards the construction of biocompatible scaffolds and the determination of the most favorable microenvironment, which will better support cell proliferation and differentiation. Perfusion bioreactors are attracting growing attention as an effective, modern tool in tissue engineering. A natural biomaterial extensively used in regenerative medicine with outstanding biocompatibility, biodegradability and non-toxic characteristics, is collagen, a structural protein with undisputed beneficial characteristics. This is a study designed according to the above considerations. 3D printed polycaprolactone (PCL) scaffolds with rectangular pores were coated with collagen either as a coating on the scaffold's trabeculae, or as a gel-cell solution penetrating scaffolds' pores. We employed histological, molecular and imaging techniques to analyze colonization, proliferation and chondrogenic differentiation of Adipose Derived Mesenchymal Stem Cells (ADMSCs). Two different differentiation culture media were employed to test chondrogenic differentiation on gelated and non gelated PCL scaffolds in static and in perfusion bioreactors dynamic culture conditions. In dynamic culture, non gelated scaffolds combined with our in house TGF-ß2 based medium, augmented chondrogenic differentiation performance, which overall was significantly less favorable compared to StemPro™ propriety medium. The beneficial mechanical stimulus of dynamic culture, appears to outgrow the disadvantage of the "weaker" TGF-ß2 medium used for chondrogenic differentiation. Even though cells in static culture grew well on the scaffold, there was limited penetration inside the construct, so the purpose of the 3D culture was not fully served. In contrast dynamic culture achieved better penetration and uniform distribution of the cells within the scaffold.

Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant.

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.

Neural control of body-plan axis in regenerating planaria.

Control of axial polarity during regeneration is a crucial open question. We developed a quantitative model of regenerating planaria, which elucidates self-assembly mechanisms of morphogen gradients required for robust body-plan control. The computational model has been developed to predict the fraction of heteromorphoses expected in a population of regenerating planaria fragments subjected to different treatments, and for fragments originating from different regions along the anterior-posterior and medio-lateral axis. This allows for a direct comparison between computational and experimental regeneration outcomes. Vector transport of morphogens was identified as a fundamental requirement to account for virtually scale-free self-assembly of the morphogen gradients observed in planarian homeostasis and regeneration. The model correctly describes altered body-plans following many known experimental manipulations, and accurately predicts outcomes of novel cutting scenarios, which we tested. We show that the vector transport field coincides with the alignment of nerve axons distributed throughout the planarian tissue, and demonstrate that the head-tail axis is controlled by the net polarity of neurons in a regenerating fragment. This model provides a comprehensive framework for mechanistically understanding fundamental aspects of body-plan regulation, and sheds new light on the role of the nervous system in directing growth and form.

In vivo Toxicity Assessment of Silver Nanoparticles in Homeostatic versus Regenerating Planarians.

Silver nanoparticles (AgNPs) belong to the most commercialized nanomaterials, used in both consumer products and medical applications. Despite its omnipresence, in-depth knowledge on the potential toxicity of nanosilver is still lacking, especially for developing organisms. Research on vertebrates is limited due to ethical concerns, and planarians are an ideal invertebrate model to study the effects of AgNPs on stem cells and developing tissues in vivo, as regeneration mimics development by triggering massive stem cell proliferation. Our results revealed a strong interference of AgNPs with tissue- and neuroregeneration which was related to an altered stem cell cycle. The presence of a PVP-coating significantly influenced toxicity outcomes, leading to elevated DNA-damage and decreased stem cell proliferation. Non-coated AgNPs had an inhibiting effect on stem cell and early progeny numbers. Overall, regenerating tissues were more sensitive to AgNP toxicity, and careful handling and appropriate decision making is needed in AgNP applications for healing and developing tissues. We emphasize on the importance of AgNP characterization, as we showed that changes in physicochemical properties influence toxicity.

Activities of MSCs Derived from Transgenic Mice Seeded on ADM Scaffolds in Wound Healing and Assessment by Advanced Optical Techniques.

BACKGROUND/AIMS: Bone marrow Mesenchymal stem cells (MSCs) are promising for promoting cutaneous wound healing through reinforcing cellular processes. We evaluated the effect of GFP-tagged MSCs transplantation on skin regeneration in excisional wounds in mice. METHODS: MSCs from GFP-labeled transgenic mice were co-cultured with acellular dermal matrix (ADM) scaffolds, and MSC-ADM scaffolds were transplanted into surgical skin wounds of BALB/c mice. After implantation, the survival and behavior of MSCs were examined by second harmonic generation and two-photon excitation fluorescence imaging, western blotting and DNA amplification and sequencing. RESULTS: GFP-tagged MSCs were retained inside the regenerating skin until day 14 post-transplantation. Alpha-smooth muscle actin (α-SMA) and vimentin (VIM) were detected at 3, 5, 7, and 14 days post-transplantation by immunofluorescence double labeling. Although the GFP+/α-SMA+- and GFP+/VIM+-cell numbers decreased gradually with healing time, α-SMA+- and VIM+-cell numbers significantly increased, most of them were endogenous functional cells which were related to angiogenesis and collagen fiber structural remodeling. CONCLUSION: Therefore, in the initial stage of wound healing, transplanted MSCs differentiated into functional cells and played paracrine roles to recruit more endogenous cells for tissue remodeling. With the disappearance of exogenous cells, endogenous cells were responsible for the latter stage of cutaneous wound healing.

First Insights into Human Fingertip Regeneration by Echo-Doppler Imaging and Wound Microenvironment Assessment.

Fingertip response to trauma represents a fascinating example of tissue regeneration. Regeneration derives from proliferative mesenchymal cells (blastema) that subsequently differentiate into soft and skeletal tissues. Clinically, conservative treatment of the amputated fingertip under occlusive dressing can shift the response to tissue loss from a wound repair process towards regeneration. When analyzing by Immunoassay the wound exudate from occlusive dressings, the concentrations of brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were higher in fingertip exudates than in burn wounds (used as controls for wound repair versus regeneration). Vascular endothelial growth factor A (VEGF-A) and platelet-derived growth factor (PDGF) were highly expressed in both samples in comparable levels. In our study, pro-inflammatory cytokines were relatively higher expressed in regenerative fingertips than in the burn wound exudates while chemokines were present in lower levels. Functional, vascular and mechanical properties of the regenerated fingertips were analyzed three months after trauma and the data were compared to the corresponding fingertip on the collateral uninjured side. While sensory recovery and morphology (pulp thickness and texture) were similar to uninjured sides, mechanical parameters (elasticity, vascularization) were increased in the regenerated fingertips. Further studies should be done to clarify the importance of inflammatory cells, immunity and growth factors in determining the outcome of the regenerative process and its influence on the clinical outcome.

Breeding Strategy Determines Rupture Incidence in Post-Infarct Healing WARPing Cardiovascular Research.

BACKGROUND: Von Willebrand A domain Related Protein (WARP), is a recently identified extracellular matrix protein. Based upon its involvement in matrix biology and its expression in the heart, we hypothesized that WARP regulates cardiac remodeling processes in the post-infarct healing process. METHODS AND RESULTS: In the mouse model of myocardial infarction (MI), WARP expression increased in the infarcted area 3-days post-MI. In the healthy myocardium WARP localized with perlecan in the basement membrane, which was disrupted upon injury. In vitro studies showed high expression of WARP by cardiac fibroblasts, which further increases upon TGFß stimulation. Furthermore, WARP expression correlated with aSMA and COL1 expression, markers of fibroblast to myofibroblast transition, in vivo and in vitro. Finally, WARP knockdown in vitro affected extra- and intracellular basic fibroblast growth factor production in myofibroblasts. To investigate the function for WARP in infarction healing, we performed an MI study in WARP knockout (KO) mice backcrossed more than 10 times on an Australian C57Bl/6-J background and bred in-house, and compared to wild type (WT) mice of the same C57Bl/6-J strain but of commercial European origin. WARP KO mice showed no mortality after MI, whereas 40% of the WT mice died due to cardiac rupture. However, when WARP KO mice were backcrossed on the European C57Bl/6-J background and bred heterozygous in-house, the previously seen protective effect in the WARP KO mice after MI was lost. Importantly, comparison of the cardiac response post-MI in WT mice bred heterozygous in-house versus commercially purchased WT mice revealed differences in cardiac rupture. CONCLUSION: These data demonstrate a redundant role for WARP in the wound healing process after MI but demonstrate that the continental/breeding/housing origin of mice of the same C57Bl6-J strain is critical in determining the susceptibility to cardiac rupture and stress the importance of using the correct littermate controls.

Shoot organogenesis from root-derived callus of Rhinacanthus nasutus (L.) Kurz. and assessment of clonal fidelity of micropropagted plants using RAPD analysis.

An efficient regeneration system was established for an ethnomedicinal shrub Rhinacanthus nasutus from root-derived callus organogenesis. The root segments were cultured on MS medium supplemented with various concentrations of Kn (1.0-4.0 µM) alone or in combination with IBA (0.2-0.6 µM) or 2, 4-D (0.5-1.5 µM). The optimum frequency (94%) of callus induction was recorded on MS medium supplemented with 3.0 µM Kn and 0.4 µM IBA. For shoot regeneration from callus, MS medium supplemented with different concentrations (1.0-7.0 µM) of BA or TDZ alone or in combination with NAA (0.2-1.0 µm) was employed. The highest frequency of shoot regeneration (91%) and mean number of shoots (28.3) were observed on MS medium supplemented with 5.0 µM BA and 0.7 µM NAA. The shoots were excised and cultured on MS medium with 4.0 µM IBA produced 3.4 roots per shoot in 88% cultures. Of the 65 plants transferred to soil 54 survived (83%). The plants were transferred to field after successful hardening. RAPD analysis of the regenerated plants showed high similarity with the mother plant.

Stability of potato (Solanum tuberosum L.) plants regenerated via somatic embryos, axillary bud proliferated shoots, microtubers and true potato seeds: a comparative phenotypic, cytogenetic and molecular assessment.

The stability, both genetic and phenotypic, of potato (Solanum tuberosum L.) cultivar Desiree plants derived from alternative propagation methodologies has been compared. Plants obtained through three clonal propagation routes-axillary-bud-proliferation, microtuberisation and a novel somatic embryogenesis system, and through true potato seeds (TPS) produced by selfing were evaluated at three levels: gross phenotype and minituber yield, changes in ploidy (measured by flow cytometry) and by molecular marker analysis [measured using AFLP (amplified fragment length polymorphism)]. The clonally propagated plants exhibited no phenotypic variation while the TPS-derived plants showed obvious phenotypic segregation. Significant differences were observed with respect to minituber yield while average plant height, at the time of harvesting, was not significantly different among plants propagated through four different routes. None of the plant types varied with respect to gross genome constitution as assessed by flow cytometry. However, a very low level of AFLP marker profile variation was seen amongst the somatic embryo (3 out of 451 bands) and microtuber (2 out of 451 bands) derived plants. Intriguingly, only AFLP markers generated using methylation sensitive restriction enzymes were found to show polymorphism. No polymorphism was observed in plants regenerated through axillary-bud-proliferation. The low level of molecular variation observed could be significant on a genome-wide scale, and is discussed in the context of possible methylation changes occurring during the process of somatic embryogenesis.

Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. Using RAPD, biochemical and morphological analysis.

Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.