Callus Response Research Articles

In vitro development of embryogenic calli and embryogenic stages in suspension cultures of mangosteen (Garcinia mangostana L.)

In this study, the potential of uncoated and coated mangosteen seed explants in forming embryogenic callus were examined in the basal Linsmaier and Skoog medium supplemented with different auxins at various concentrations. Among the highest percentage of callus response (93.3%) was obtained when uncoated seed explants were cultured in basal LS medium containing 8 mg/L 2,4-D. Combining of cytokinins and 2,4-D to improve embryogenicity of calli showed that among the highest percentage of callus response (80%) and the lowest percentage of callus browning (53.53%) with yellowish and compact nodular calli were obtained on MS medum supplemented with 8 mg/L 2,4-D and 0.1 mg/L BAP. Addition of glutamine into MS medium containing 8 and 0.1 mg/L BAP performed that glutamine did not increase the growth of calli, however texture (friable) and color callus (yellowish) was improved. The growth and multiplication of cells in suspension cultures showed that the cells were able to divide and proliferate even though cultured in half strength MS liquid medium without 2,4-D. After six months of culture, the heart embryogenic stage was obtained only on medium supplemented with 1 mg/L BAP. The globular and torpedo embryogenic stages were obtained on media supplemented with 1, 3 and 9 mg/L TDZ after five months of culture. Key words: Mangosteen, embryogenic callus, suspension culture, in vitro.

Responses of distraction regenerate to high-frequency traction at a rapid rate

Continuous traction is capable of creating an optimal biological environment for bone healing which may finally compensate for the rapid distraction rate in distraction osteogenesis. This study was designed to investigate the response of distraction callus to continuous distraction at a rapid rate using a rabbit model of mandibular lengthening. Thirty adult New Zealand white rabbits were randomly assigned to the intermittent (1 step/d) or continuous distraction (8 steps/s) group, with 15 in each. After osteotomy, manual-driven or autodriven distractor was adapted accordingly. The distraction was activated at a rate of 3.0 mm/d for 4 days. Five rabbits in each group were killed at week 2, week 4, and week 12 of consolidation, respectively. Plain radiography, microcomputed tomography, and histology examinations were used to evaluate the bone regeneration status. Plain radiographs and histologic studies demonstrated more advanced bone healing in continuous distraction group than that in intermittent distraction group at all the examined time points. Quantitative microcomputed tomography analysis showed significantly higher bone volume in continuous distraction group at week 2 (p < 0.01) and week 4 (p < 0.05) of consolidation. Continuous traction by autodriven distractor could be a promising clinical alternative to shorten the treatment course of distraction osteogenesis. Further studies to test its clinical potential using large animals that have similar metabolic rate and muscular resistance with human being are necessary.

Endogenous auxin level is a critical determinant for in vitro adventitious shoot regeneration in potato (Solanum tuberosum L.)

Endogenous levels of indole-3-acetic acid (IAA) were determined in the internodal explants of Indian tetraploid potato cultivars (cvs) viz., Kufri Sutlej (K.Sutlej) and Kufri Giriraj (K.Giriraj). Seven fold higher level of endogenous IAA was recorded for cv K.Sutlej over cv K.Giriraj. As a result, perhaps there was a callusing response from the cut end of the internodal explants of both the cvs in the MS basal medium. The extent of callusing was relatively higher in cv K.Sutlej when compared to that in K.Giriraj. The callusing response was inhibitory to shoot morphogenesis. Inclusion of an established anti-auxin, 2,3,5-tri-iodobenzoic acid (TIBA) in the regeneration medium facilitated a high frequency adventitious shoot regeneration response with lower cytokinin levels. Murashige and Skoog (MS) medium containing TIBA at 2.5 mg l−1 and 0.25 mg l−1 zeatin evoked a 100% regeneration response (4.5 shoot buds per explant) in cv K.Sutlej within 20–25 days. However, in cv K.Giriraj, which had lower levels of endogenous IAA, 80% regeneration response (1.4 shoot buds per explant) was recorded in an extended period of 40–45 days on a medium containing 0.5 mg l−1 TIBA and 0.1 mg l−1 zeatin. Although, TIBA and zeatin induced shoot bud formation, it failed to support sustained growth of the regenerated shoots in cv K.Giriraj. Hence, 0.01 mg l−1 α-naphthaleneacetic acid (NAA) with a relatively higher concentration of zeatin (1.0 mg l−1) were used for sustained shoot regeneration (3.3 shoot buds per explant) within 25 days. From our results, it is evident that there was a difference in the requirement of exogenous zeatin levels required to induce a regeneration response in two cultivars of potato and this is attributed to the variable levels of endogenous IAA.

Propagation and Effects of the Pinewood Nematode on Calli of Various Plants

Bursaphelenchus xylophilus and its closely related species, B. mucronatus were successfully propagated well on calli of tobacco (Nicotiana tabacum), alfalfa (Medicago sativa) and several pine species. Different propagation rates or different maximum populations were recorded. Most nematodes occurred in the outer layer of the callus, in which cells appeared to be dead. Tannin accumulated in the nematode- inoculated callus tissues. The correlation of the tannin content with the number of Many studies have been done on pine wilt disease caused by the pinewood nematode, Bursaphelenchus xylophilus (STEINER and BUHRER) NICKLE. Several of these studies have focused on host resistance and on chemical components of the host that attract nematodes or promote their reproduction3' 15) as well as factors that inhibit invasion of host tissue3) or affect nematode mobilityn. FUTAI and FURUNO7 who inoculated the pinewood nematodes into young trees of 30 pine species, showed that resistance to the pinewood nematode varies among pine species. They correlated the differences in host resistance to taxonomic categories of pines (sensu CLITCHFIELD and LITTLE, 1966), but could not explain the cause of such differences. The mechanisms of pine wilt and host resistance have been studied by propagating B. xylophilus on tissue cultures of various plants. For example, TAMURA and MAMIYA13, 14) observed that B. xylophilus feeds and reproduces on the calli of Pinus densiflora and P. thunbergii, both of which are susceptible to pine wilt. TAMURA and DROPKINI1) propagated B. xylophilus on the callus of P. jeffreyi which is a resistant species to pine wilt. B. xylophilus has been propagated on calli of non-host plants such as alfalfa (Medicago sativa)12) and Japanese red cedar (Cryptomeria japonica). It is also well-known that B. xylophilus can be propagated on various fungi. These examples demonstrate the nematode's ability to feed and reproduce on various kinds of plant callus tissues and fungal cells. In the present study, the population buildup of B. xylophilus and that of non-pathogenic related species B. mucronatus were compared on calli of four pine species and alfalfa. In addition, the reproduction of B. xylophilus was also examined on calli of the F1 hybrid of P. thunbergii x P. massoniana and N. tabacum. Special consideration was also paid to the effect of nematode feeding and population buildup on callus responses, and to the way this relates to resistance shown in nursery trees. Tannin production in inoculated callus was also determined, which is known to be associated with host resistance to the pinewood nematode6). Nematode feeding and movement on callus cultures was also observed microscopically.

Proteomic changes in grape embryogenic callus in response to Agrobacterium tumefaciens-mediated transformation

Agrobacterium tumefaciens-mediated transformation is highly required for studies of grapevine gene function and of huge potential for tailored variety improvements. However, grape is recalcitrant to transformation, and the underlying mechanism is largely unknown. To better understand the overall response of grapevine to A. tumefaciens-mediated transformation, the proteomic profile of cv. Prime embryogenic callus (EC) after co-cultivation with A. tumefaciens was investigated by two-dimensional electrophoresis and MALDI–TOF–MS analysis. Over 1100 protein spots were detected in both inoculated and control EC, 69 of which showed significantly differential expression; 38 of these were successfully identified. The proteins significantly up-regulated 3 d after inoculation were PR10, resistance protein Pto, secretory peroxidase, cinnamoyl-CoA reductase and different expression regulators; down-regulated proteins were ascorbate peroxidase, tocopherol cyclase, Hsp 70 and proteins involved in the ubiquitin-associated protein-degradation pathway. A. tumefaciens transformation-induced oxidative burst and modified protein-degradation pathways were further validated with biochemical measurements. Our results reveal that agrobacterial transformation markedly inhibits the cellular ROS-removal system, mitochondrial energy metabolism and the protein-degradation machinery for misfolded proteins, while the apoptosis signaling pathway and hypersensitive response are strengthened, which might partially explain the low efficiency and severe EC necrosis in grape transformation.

Callus culture and plant regeneration from seedling explants in ‘Poshita’ indian ginseng

The study was conducted to establish callus culture and plant regeneration in Withania somnifera (L.) Dunal. cv. Poshita with different explants and different auxin types and concentrations. Nodal explant showed better callusing response irrespective of auxin concentrations supplemented in the media, as compared to epicotyls, hypocotyls, leaves, and root tips. Maximum callusing score of 4.28 was recorded when nodes were cultured on Murashige and Skoog’s (MS) media containing 2 mg · L−1 naphthaleneacetic acid (NAA). Root tip and hypocotyls explants, which were found poor in inducing callus, showed highest proliferation of 6.57 and 6.00, respectively, on media with 2,4-Dichlorophenoxyacetic acid (2,4-D) for subculture. Highest number of shoots (16.0) per explant was obtained from epicotyls and nodes cultured on 1 and 2 mg · L−1 benzyladenine (BA), respectively. Indole-3-butyric acid (IBA) was found to be superior source of auxin for all rooting parameters, except percent rooting. Number of primary roots was found maximum (24.8) with 2.0 mg · L−1 IBA. Irrespective of auxin used, the length of primary root decreased with increase in concentration, maximum (6.18 cm) being in media with 0.5 mg · L−1 IBA. Plantlets were best hardened when agropeat was used as a substrate.

Influence of different sterilization methods on callus initiation and production of pigmented callus in Arnebia densiflora Ledeb.

We analyzed the effects of sodium hypochlorite and Plant Preservative Mixture (PPM) on surface sterilization. We also examined the effects that the addition of an antibiotic-antimycotic solution to the culture medium had on callus induction. Explants were initially sterilized with different concentrations of sodium hypochlorite and cultured on MS media containing kinetin (0.29 \muM) and naphthalene acetic acid (NAA, 10 \muM). No calluses were produced, either because of contamination of the explants, or loss of explants as a result of the high levels of sodium hypochlorite. The application of PPM and antibiotics at different concentrations reduced contamination and led to callus induction from shoot apexes and young root explants. The best callus responses were obtained using PPM at 1%-2%, whereas callus induction on shoot apexes diminished at higher concentrations (4% PPM). This is the first report of successful sterilization and reduced contamination of explants from naturally field grown A. densiflora by PPM. Moreover, established callus cultures produced pigmented calluses, which were analyzed spectrophotometrically.

α1 adrenergic receptor agonist, phenylephrine, actively contracts early rat rib fracture callus ex vivo

Early, soft fracture callus that links fracture ends together is smooth muscle-like in nature. We aimed to determine if early fracture callus could be induced to contract and relax ex vivo by similar pathways to smooth muscle, that is, contraction via α(1) adrenergic receptor (α(1) AR) activation with phenylephrine (PE) and relaxation via β(2) adrenergic receptor (β(2) AR) stimulation with terbutaline. A sensitive force transducer quantified 7 day rat rib fracture callus responses in modified Krebs-Henseliet (KH) solutions. Unfractured ribs along with 7, 14, and 21 day fracture calluses were analyzed for both α(1) AR and β(2) AR gene expression using qPCR, whilst 7 day fracture callus was examined via immunohistochemistry for both α(1) AR and β(2) AR- immunoreactivity. In 7 day callus, PE (10(-6) M) significantly induced an increase in force that was greater than passive force generated in calcium-free KH (n = 8, mean 51% increase, 95% CI: 26-76%). PE-induced contractions in calluses were attenuated by the α(1) AR antagonist, prazosin (10(-6) M; n = 7, mean 5% increase, 95% CI: 2-11%). Terbutaline did not relax callus. Gene expression of α(1) ARs was constant throughout fracture healing; however, β(2) AR expression was down-regulated at 7 days compared to unfractured rib (p < 0.01). Furthermore, osteoprogenitor cells of early fibrous callus displayed considerable α(1) AR-like immunoreactivity but not β(2) AR-like immunoreactivity. Here, we demonstrate for the first time that early fracture callus can be pharmacologically induced to contract. We propose that increased concentrations of α(1) AR agonists such as noradrenaline may tonically contract callus in vivo to promote osteogenesis.

Plant regeneration using immature zygotic embryos of Tribulus terrestris

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.

FIRST REPORT ON CALLUS INDUCTION IN ASHOKA (SARACA INDICA): AN IMPORTANT MEDICINAL PLANT

Leaves cultured on Murashige and Skoog (MS) media supplemented with BAP (2 mg/L) and NAA (0.5 mg/L) showed calli formation in 29.4% explants. When cultured on half strength MS media (2 mg/L BAP and 0.5 mg/L NAA), 13.3% explants could produce calli. Hypocotyls segments cultured on full strength MS media showed good callusing response (52.6%). Culture of explants on the half strength MS media produced calli in 77.8% of explants. When segments of epicotyls were placed on full strength MS media, a callusing response of 83.3% was observed. Segments when were placed on half strength MS media, a maximum response of 88.9% was observed. The calli failed to differentiate into shoots when sub-cultured on shoot induction media.

A METHOD TO IMPROVE THE INDUCTION OF CALLUS FROM SAFFRON (CROCUS SATIVUS LINNEO) CORMS

In vitro callus and suspended cells induction's response was compared from saffron corms (Crocus sativus L.) from a Spanish cultivar; some corms were immediately cultivated in vitro in November 2006 and the others were preserved in distilled water in laboratory until daughter corms formed and in vitro cultured in May 2007. For the explants of the mother corms, the medium used was Murashige-Skoog (MS), with 2-4 dichlorophenoxy acetic acid (2,4-D, 0, 1, 2 and 4 mg.L -1 ) and kinetin (Kin., 0, 1 mg.L -1 ) in liquid medium and in agar gelified medium. The explants of daughter corms had the same treatments and they were planted in MS medium with Naphtalene Acetic Acid (NAA, 0, 1, 2 and 4 mg.L -1 ) and Kin (0, 1 mg.L -1 ), as well in gel as liquid media. In mother corms in gel the average induction time was 31.6 days with 24% induction frequency and 85% contamination rate; in liquid medium the contamination reached 100%. In the case of daughter corms obtained in laboratory, treatments in gel had an average induction time of 12.4 days and 100% induction frequency and 15% contamination; in liquid treatments the appearance time of free cells varied from 10 to 14 days with 100% induction frequency and 25% contamination rate; there was no significant difference (P>0.1) among the different combinations with auxins, in gel as well as liquid media, in whose formulae there was always callus or free cells, whereas in the case of the media with kinetin only and witnesses there was no response at all. However, there was significant difference (P<0.045) between treatments in gel and those of the liquid medium. Additionally, whole daughter corms were planted both in gel and liquid media with NAA 2 mg.L -1 and Kin 1 mg.L -1 , where induction of callus in gel medium was observed as well above liquid medium level and suspended cells within liquid medium.

Callus induction and whole plant regeneration in elite Indian maize (Zea mays L.) inbreds

Callus induction and regeneration ability of five elite maize inbred lines, CM 111, CM 117, CM 124, CM 125 and CM 300 were investigated using 14-day-old immature embryos as explants. Genotype, medium, source of auxin and their concentrations influenced induction of callus. Explants grown on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid at 1 mg l−1 showed the highest frequency of callusing. Among all the media tested, explants grown on N6 medium gave the highest frequency of organogenic callus. Moreover, N6 supplemented with Dicamba promoted higher callus response in terms of both frequency of induction as well as quality, compared to N6 medium with 2,4-D. N6 supplemented with 2 mg l−1 Dicamba induced the highest frequency of organogenic callus. Among the five genotypes tested, CM 124, CM 125, and CM 300 gave the best callus. Explants of both CM 124 and CM 300 incubated on MS medium supplemented with 1 mg l−1 benzyladenine and 0.5 mg l−1 indole acetic acid promoted the highest frequency of shoot induction. Though CM 124 induced higher percentage of shoot formation than CM 300, the mean number of developed shoots per explant was higher for CM 300. The highest frequency of root formation was observed when shoots were grown on MS medium supplemented with 2 mg l−1 naphathalene acetic acid. Percentage of regenerated plants ranged from 54 to 66.

In vitro callus initiation of a 'threatened' Nigerian leafy vegetable, Gnetum africanum (WILW)

Gnetum africanum is a green leafy vegetable found in Nigeria, where it is a highly valued food source. Stocks of this plant in the wild are increasingly threatened by land transformation and harvesting methods are unsustainable. In vitro callus initiation of G. africanum has been developed. The surfaces of 3 to 4 day old leaf explants were sterilized before exposure to a range of different concentrations of plant growth regulators. In vitro callus initiation of explants in single concentrations of auxins (2,4D: 2-4-dichloroxyphenoxyacetic and pichloram) did not initiate callus after 3 months of culture. However, combinations of cytokines (BAP: 6-benzylaminopurine, and Kinetin) and auxins initiated callus. The highest percentage callus initiation response of 100% was observed in the combination of BAP (1.0 mg/l) + 2,4D (7 mg/l). There was a significant difference (P 0.05) between the degree of callus response and callus size to these different media. Key words: BAP (6-benzylaminopurine), callus initiation, Gnetum africanum, kinetin, 2,4D (2-4-dichloroxyphenoxyacetic), pichloram.

In-vitro callus induction and shoot regeneration in Ipomoea obscura (L.): potent Indian medicinal plant

Callus of the leaves, node and bud of Ipomoea obscura (L) was initiated on MS basal media supplemented with various combinations of auxins 2,4- dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid with cytokines kinetin and benzyl amino-purin (BAP). Callus initiation was observed in all media but with varied mass. Highest percentage of callus response was obtained in combination of 2,4-D (0.8 mg/L) with kinetin (0.8mg/L); NAA (0.2mg/L) with BAP(0.2 mg/L); NAA (0.8 mg/L) with kinetin (0.8 mg/L) for explants leaves, node and bud, respectively. The callus was white in 2,4-D- and green in NAA- supplemented MS media. An efficient micropropogation protocol was developed for medicinal plant Ipomoea obscura (L.) by in-vitro culture of nodal part of mature plant. The MS media supplemented with 0.8 mg/L NAA with 0.8 mg/L kinetin induced three shoots per node in an average and was best for axillary bud proliferation. Keywords: Ipomoea obscura, tissue culture, medicinal plant

In-vitro Callus Induction and Shoot Regeneration in Ipomoea obscura (l.): Potent Indian Medicinal Plant

Callus of the leaves, node and bud of Ipomoea obscura (L) was initiated on MS basal media supplemented with various combinations of auxins 2,4- dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid with cytokines kinetin and benzyl amino-purin (BAP). Callus initiation was observed in all media but with varied mass. Highest percentage of callus response was obtained in combination of 2,4-D (0.8 mg/L) with kinetin (0.8mg/L); NAA (0.2mg/L) with BAP(0.2 mg/L); NAA (0.8 mg/L) with kinetin (0.8 mg/L) for explants leaves, node and bud, respectively. The callus was white in 2,4-D- and green in NAA- supplemented MS media. An efficient micropropogation protocol was developed for medicinal plant Ipomoea obscura (L.) by in-vitro culture of nodal part of mature plant. The MS media supplemented with 0.8 mg/L NAA with 0.8 mg/L kinetin induced three shoots per node in an average and was best for axillary bud proliferation.

SELECTION OF SALT TOLERANT CLONES OF SOME MEDICINAL PLANTS THROUGH TISSUE CULTURE TECHNIQUE: I. Hyoscyamus muticus L. PLANT.

Response of Egyptian henbane plant callus (Hyoscyamus muticus L.) to various levels of salt was investigated. Two tolerant clones to salt were selected from callus cultured on selection medium contained 6000 and 8000 ppm of salts, respectively. Tolerant clones were capable of proliferating and growing on media contained the same salt concentrations and supplemented with 1.5 ppm of BA. Vegetative characters were significantly enhanced in general at the lower salt concentration (6000 ppm) especially with the media containing 0.12 g/ L KH2PO4 +4 ppm kinetin + 0.5 ppm IBA. Using of MS media at its half strength with 1ppm NAA was the best for henbane root growth at 6000ppm of salt. Survival percentage reached up to 28.00 % for clone A(selected at 6000ppm ) and 32.02 % for clone B(selected at 8000ppm) with a total alkaloid percentage of 0.90 and 0.98 %, respectively .

Somatic embryogenesis and plant regeneration of northern red oak (Quercus rubra L.)

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 culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.

INDUCTION OF SOMATIC EMBRYOS AND IMPROVEMENT OF EMBRYOGENIC CALLUS BY TWO TYPES OF SILVER ON DATE PALM (PHOENIX DACTYLEFERA L.)

The response of embryogenic callus of date palm cvs. (Pertomuda and Skooty) was tested on media containing two types of silver (STS and Ag-NO3) alone or combined with 0.25 & 0.5 mg/l 2ip. Addition of STS onto callus induction medium sig-nificantly enhanced embryogenic callus production (both growth and induction). AgNO3 as a heavy metal ion made a stress treatment on callus to proliferation embryos than STS, this proliferation was increased at a low concentration of AgNO3, but in the presence of 2ip gave the opposite trend, the embryos increased at 4 mg/l or 6 mg/l AgNO3 + 0.5 mg/l 2ip and proceeded normally embryos for both Pertomuda and Skooty date palm.Abbreviations: STS = Silver thiosulphate; EC = embryogenic callus; NAA = α-naphthaleneacetic acid and 2ip = N6-2-isopentenyladenine.

Simulation of fracture healing incorporating mechanoregulation of tissue differentiation and dispersal/proliferation of cells

Modelling the course of healing of a long bone subjected to loading has been the subject of several investigations. These have succeeded in predicting the differentiation of tissues in the callus in response to a static mechanical load and the diffusion of biological factors. In this paper an approach is presented which includes both mechanoregulation of tissue differentiation and the diffusion and proliferation of cell populations (mesenchymal stem cells, fibroblasts, chondrocytes, and osteoblasts). This is achieved in a three-dimensional poroelastic finite element model which, being poroelastic, can model the effect of the frequency of dynamic loading. Given the number of parameters involved in the simulation, a parameter variation study is reported, and final parameters are selected based on comparison with an in vivo experiment. The model predicts that asymmetric loading creates an asymmetric distribution of tissues in the callus, but only for high bending moments. Furthermore the frequency of loading is predicted to have an effect. In conclusion, a numerical algorithm is presented incorporating both mechanoregulation and evolution of cell populations, and it proves capable of predicting realistic difference in bone healing in a 3D fracture callus.

The effect of seed size on tissue culture response of callus from endosperm-supported mature embryos of barley (Hordeum vulgareL.)

Although immature embryos are the most suitable explants to regenerate cereals, their limited availability throughout the year hampers frequent experimentation. In order to circumvent this problem, endosperm-supported mature embryos, which are available throughout the year, are proposed for utilization in cereal tissue culture studies. In the present study, the effect of seed size on in vitro seed germination, seedling growth, callus induction and plant regeneration, as well as the relationships between these parameters, were investigated in three barley (Hordeum vulgare L.) genotypes. Seeds were designated as large or small for each genotype and seed germinations were achieved in Petri dishes between two sheets of pre-wetted filter paper. Germination percentages of seeds, seedling growth rates, shoot and root lengths, and seedling fresh and dry weights were examined. Mature embryos from imbibed and dehulled seeds were aseptically moved slightly with a scalpel. For callus induction, 8 mg 1−1, 2,4-D dissolved in water, and for plant regeneration, hormone-free MS medium, was utilized. Highest values with respect to seed germination percentage, seedling growth, callus induction and plant regeneration were obtained using large seeds. Rooting of regenerated shoots and plantlet recovery were also successfully achieved. Based on the significant positive correlations observed between all parameters, we concluded that cultures derived from endosperm-supported mature embryos within large seeds should be employed for achieving superior tissue culture response.