Consecutive Regeneration Research Articles

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.

Nitrate removal from water by quaternized pine bark using choline based ionic liquid analogue

AbstractBACKGROUND: The objective of this study was to quaternize pine bark (PB) wood residues using green chemistry and to use the quaternized PB to remove nitrate (NO3−) from water. The quaternization process was achieved by reacting the wood residues with an ionic liquid analogue comprised of a choline chloride derivative and urea. Batch adsorption tests were used to delineate the NO3− uptake by the modified pine bark (MPB). Fourier Transform Infrared Spectroscopy (FTIR) analysis and Zeta potential measurements were used to characterize the changes at the surface of the PB due to quaternization and NO3− uptake.RESULTS: The MPB has a maximum NO3− uptake capacity of 2.91 mmol g−1. The NO3− uptake kinetics indicated that diffusion through the boundary layer of the MPB was the rate limiting step. The Langmuir adsorption model provided a better fit for the uptake data than the Freundlich model, indicating monolayer adsorption. The uptake process was found dependent on concentration, pH and ionic strength, and was also spontaneous and exothermic. The desorption–regeneration experimental results indicated a 95% efficiency after five consecutive regeneration cycles.CONCLUSIONS: The quaternization technique was found very effective for developing effective and green anion exchange resins to remove NO3− from water. © 2012 Society of Chemical Industry

Trait variations along a regenerative chronosequence in the herb layer of submediterranean forests

The aim of this paper is to assess the functional shifts of the herb layer in the submediterranean Ostrya carpinifolia coppiced forests (central Italy) along a coppicing rotation cycle. More specifically, the following questions were addressed: i) is there a pattern in functional trait composition of the herb layer along a regeneration chronosequence?; ii) which traits states differentiate each regeneration stage?; iii) are patterns of trait state variation related to the change of the environmental conditions? Species cover percentage was recorded in 54 plots (20 m × 20 m) with homogeneous ecological conditions. Relevés, ordered on the basis of the time since the last coppicing event and grouped into three age classes, were analysed with regard to trait variation, based on species absolute and relative abundance. Differences in light, temperature, soil moisture, and nutrients bioindicator values between consecutive regeneration stages were tested using the non-parametric Mann–Whitney U-test. Multi-response permutation procedures (MRPP) revealed statistically significant separation between young and intermediate-aged stands with regard to most traits. Indicator species analysis (ISA) highlighted indicator trait states, which were filtered, along the chronosequence, by changes in environmental conditions. Redundancy analysis (RDA) revealed that light intensity had the greatest effect on traits states variation from the first to the second regeneration stage, while variation from the second to the third age classes was affected by temperature. Young stands were differentiated by short cycle species with acquisitive strategies that only propagated by sexual reproduction, with light seeds, summer green and overwintering green leaves, and a long flowering duration. Intermediate-aged and mature stands were characterized by traits associated with early leaf and flower production, high persistence in time, and showing retentive strategies aimed at resource storage (e.g., geophytes, spring green leaves, rhizomes, and mesomorphic/hygromorphic leaves).

Spermatophore cryopreservation and artificial insemination of black tiger shrimp, Penaeus monodon (Fabricius)

To develop an appropriate cryopreservation protocol for spermatophores of black tiger shrimp, Penaeus monodon, three cryoprotectants (dimethyl sulphoxide (DMSO), methanol (MeOH) and ethylene glycol (EG)) at two concentrations (5% and 10%) were examined. Artificial implantation of spermatophores was also carried out to assess the fertilizing ability of fresh and post-thaw spermatophores. Spermatophores were collected during consecutive regenerations (15-day intervals) and assessed for qualitative and quantitative changes and also for fertilizing ability by implantation. The mean fertilization rate for artificial insemination using post-thaw spermatophore was 79.9±3.7%, lower than the fertilization rates observed for artificial implantation using fresh spermatophore and natural mating. Mean hatch rates for fresh spermatophore, frozen-thawed spermatophore and natural mating were 88.8±0.6%, 87.8±0.4% and 88.3±0.5%, respectively; and there was no difference among the three groups. The mean fertilization rate of spermatophores collected during the first stripping was higher (90.6±0.6) than during the second stripping (85.7±2.6), but the mean hatch rate was not different between the two strippings. The highest mean sperm viability (79.7±0.4%) was obtained from DMSO (5%), with no survival observed in the 10% MeOH treatment. Spermatophore weight, total sperm count and percentage of abnormal sperm were not different between spermatophores collected at the first and second stripping. This is the first study to report high fertilization and hatch rates from cryopreserved spermatophore using artificial implantation of spermatophore before spawning.

Studies on genetic changes in rye samples (Secale cereale L.) maintained in a seed bank

The aim of this study was to identify genetic changes in rye seeds induced by natural ageing during long-term storage and consecutive regeneration cycles under gene bank conditions. Genomic DNA from four rye samples varying in their initial viability after one and three cycles of reproduction was analyzed by AFLP (amplified fragment length polymorphism) fingerprinting. Seven EcoRI/MseI primer combinations defined 663 fragments, and seven PstI/MseI primer combinations defined 551 fragments. The variation in the frequency of the seventy-four EcoRI/MseI bands was statistically significant between samples. These changes could be attributed to genetic changes occurring during storage and regeneration. However, the PstI/MseI fragments appeared to be uninfluenced by seed ageing, regeneration and propagation. A combined Principle Coordinate Analysis revealed differences between samples with different initial viability. We showed that materials with low initial viability differ in their response from highly viable ones, and that the changes exhibited in the former case are preserved through regeneration cycles.

Sperm Quality Over Consecutive Spermatophore Regenerations in the Pacific White Shrimp Litopenaeus vannamei

AbstractThe reproductive potential of males plays an important role in the productivity of captive penaeids. Males present a large variability in their reproductive potential, and thus the selection of appropriate males for reproduction is highly desirable. The present study compares sperm quality at the beginning of the experiment (baseline values) with variations in sperm quality as a result of consecutive spermatophore regenerations. This was done to evaluate possible predictive criteria for optimal sperm quality based on morphological and biochemical criteria in pond‐reared Pacific white shrimp Litopenaeus vannamei males. Sperm quality was similar between left and right spermatophore. Sperm count and proportion of normal sperm increased by 86 and 65% (P < 0.05) respectively, from baseline values to third spermatophore regeneration. Proportion of dead sperm progressively decreased with consecutive regenerations, attaining values 33% lower by the third regeneration, compared to baseline values. This indicates that no decrease in sperm quality occurs in L. vannamei during consecutive regenerations and that the existing spermatophores at stocking of males should be expelled to have higher sperm quality in regenerated spermatophores. Furthermore, this procedure allows selecting individuals with high baseline sperm quality based on significant correlations between baseline and regenerated sperm quality observed in the present work. Baseline values of glucose concentration in the hemolymph were positively correlated to baseline values of sperm count (r = 03, P < 0.05). In contrast, baseline values of several lipids in the hemolymph were negatively correlated to several traits of sperm quality at first sampling (baseline values) or at first regeneration. These results are discussed in terms of nutritional requirements of males and of possible predictive criteria of sperm quality.

Evanescent wave long-period fiber bragg grating as an immobilized antibody biosensor.

An immunosensor using a long-period grating (LPG) was used for sensitive detection of antibody-antigen reactions. Goat anti-human IgG (antibody) was immobilized on the surface of the LPG, and detection of specific antibody-antigen binding was investigated. This sensor operates using total internal reflection where an evanescent field interacts with bound antibody immobilized over the grating region. The reaction between antibody and antigen altered the LPG transmission spectrum and was monitored in real time as a change in refractive index, thereby eliminating the need for labeling antigen molecules. Human IgG binding was observed to be concentration dependent over a range of 2-100 microg mL-1, and equilibrium bound antigen levels could be attained in approximately 5 min using an initial rate determination. Binding specificity was confirmed using human interleukin-2 and bovine serum albumin as controls, and nonspecific adsorption of proteins did not significantly interfere with detection of binding. Antigen detection in a heterogeneous protein mixture and in crude cell lysate from Escherichia coli was also confirmed. Moreover, regeneration of the LPG surface via diethylamine treatment resulted in approximately 80% removal of bound antigen. Subsequently, fibers reexposed to antigen retained greater than 85% of the initial signal after five consecutive regeneration cycles.

Regeneration of immobilized antibodies on fiber optic probes

The regeneration of antibodies covalently immobilized to an optical fibre surface was investigated by dissociation of the antibody-antigen complex with three different solvents: (a) an acidic solution (0·1 M glycine hydrochloride in 50% (v/v) ethylene glycol, pH 1·75), (b) a basic solution (0·05 M tetraethylamine in 50% (v/v) ethylene glycol, pH 11·0) and (c) 50% (v/v) ethanol in PBS. The fibres coated with polyclonal rabbit anti-goat antibody against a large protein retained 70% and 65% of the original signal after five consecutive regenerations with acidic and basic solvent systems, respectively. The fibres coated with monoclonal mouse anti-trinitrobenzene antibody specific for a small organic molecule, retained over 90% of the original signal when regenerated with basic and ethanol solutions. This study evaluated regeneration and reuse of antibody-coated fibre optic biosensors as a means of reducing routine laboratory analysis costs and time.

Peripheral nerve allografts: survey of present state in an experimental model of the rat.

Rejection and regeneration processes in peripheral nerve allografts are analyzed in this review of a series of experiments with special reference to the possible clinical application of peripheral nerve allografting in clinical reconstructive surgery. A long segment of the sciatic nerve (2.5 cm) was grafted between congenic rat strains across a maximal genetic barrier; immunohistologically, donor- and recipient-derived structures can be differentiated. If allografting was performed without immunosuppression, a rejection response with consecutive regeneration of minor quality was observed. Under immunosuppression with cyclosporin A no rejection response was observed and regeneration quality was comparable to control autografts. The persistence of donor-derived Schwann cells in the immunosuppressed allografts can be demonstrated immunohistologically. After discontinuation of immunosuppression a rejection response is exerted. We conclude that Schwann cells are eliminated from peripheral nerves during rejection. Consecutive regeneration of minor quality is possible, which implies the ingrowth of recipient-derived Schwann cells into the rejected allograft. Under immunosuppression, allogenic Schwann cells survive and actively promote regeneration. They are still immunologically competent and can exert rejection when immunosuppression is discontinued. A certain degree of replacement of donor-derived Schwann cells seems possible.

Time course of alterations in muscle transfers with microneurovascular anastomoses. An experimental study in the rectus femoris muscle of the rabbit.

The time course of alterations in muscle transfers with microneurovascular anastomoses was studied in 17 rabbits. The left rectus femoris muscle was transferred to the right side. For comparison, in some animals the right rectus femoris muscle was transferred from the right to the left side, but without vascular repair. Two, seven, 14, 21, and 30 days after transfer, the electric excitability, macroscopic appearance, histology, histochemistry, ultrastructure, and activity of muscle enzymes were assessed. In the transfers with microneurovascular anastomoses, almost all muscle fibers survived. Alterations were limited to those typical of a denervation-reinnervation process. Contrary to this, only a few atrophic fibers survived in the periphery of the transfers without vascular repair. By far, the greater central part underwent necrosis. A considerable amount of connective tissue developed. These results clearly show the functional superiority of microsurgically vascularized muscle transfers over those without vascular anastomoses. Excellent functional recovery is possible because the process of complete degeneration and consecutive regeneration, with inevitable augmentation of connective tissue, is prevented by the performance of vascular anastomoses.