Callus Tissue Formation Research Articles

Mechanical Load Modulates the Stimulatory Effect of BMP2 in a Rat Nonunion Model

Local application of bone morphogenetic proteins (BMPs) at the fracture site is known to stimulate bone regeneration. However, recent studies illustrate that the BMP-initiated mineralization may be enhanced by additional mechanical stimulation. Therefore, bone healing was monitored in vivo in order to investigate the effect of mechanical loading on the initiation and maturation of mineralization after cytokine treatment. We hypothesized that the mechanical stimulation would further enhance the efficacy of BMP2 treatment. Female Sprague-Dawley rats underwent a 5-mm defect, stabilized with an external fixator. Type I collagen scaffolds containing 50 μg of BMP2 diluted in a solvent or solvent only were placed into the defects. The BMP2-treated specimens and control specimens were then each divided into two groups: one that underwent mechanical loading and a nonloaded group. In vivo loading began immediately after surgery and continued once per week for the entire 6-week experimental period. For all groups, the newly formed callus tissue was quantitatively evaluated first by in vivo microcomputed tomography at 2, 4, and 6 weeks and further by histologic or histomorphometric analysis at 6 weeks postoperation. Mechanical stimulation with BMP2 treatment significantly enhanced mineralized tissue volume and mineral content at 2 weeks. Histological analysis demonstrated a significantly greater area of fibrous connective tissue including bone marrow in the stimulated group, suggesting reconstitution of the endosteal canal and more advanced bone remodeling present in the mechanical loaded group. Both groups receiving BMP2 underwent massive bone formation, achieving bony bridging after only 2 weeks, while both control groups, receiving solvent only, revealed a persisting nonunion, filled with fibrous connective tissue, prolapsed muscle tissue, and a sealed medullary canal at week 6. Mechanical loading further enhanced the efficacy of BMP2 application evidenced by increased mineralized tissue volume and mineralization at the stage of bony callus bridging. These data suggest that already a minimal amount of mechanical stimulation through load bearing or exercise may be a promising adjunct stimulus to enhance the efficacy of cytokine treatment in segmental defects. Further studies are required to elucidate the mechanistic interplay between mechanical and biological stimuli.

Acute Alcohol Exposure Impairs Fracture Healing and Deregulates β‐Catenin Signaling in the Fracture Callus

Alcohol abuse is a risk factor for bone damage and fracture-related complications. Through precise β-catenin signaling, canonical Wnt signaling plays a key role in fracture repair by promoting the differentiation of new bone and cartilage cells. In this study, we examined the effects of alcohol on the Wnt pathway in injured bone using a murine model of alcohol-induced impaired fracture healing. Male C57Bl/6 or T cell factor (TCF)-transgenic mice were administered 3 daily intraperitoneal doses of alcohol or saline. One hour following the final injection, mice were subjected to a stabilized, mid-shaft tibial fracture. Injured and contralateral tibias were harvested at 6, 9, or 14days post-fracture for the analysis of biomechanical strength, callus tissue composition, and Wnt/β-catenin signaling. Acute alcohol treatment was associated with a significant decrease in fracture callus volume, diameter, and biomechanical strength at day 14 post-fracture. Histology revealed an alcohol-related reduction in cartilage and bone formation at the fracture site, and that alcohol inhibited normal cartilage maturation. Acute alcohol exposure caused a significant 2.3-fold increase in total β-catenin protein at day 6 and a significant decrease of 53 and 56% at days 9 and 14, respectively. lacZ staining in β-galactosidase-expressing TCF-transgenic mice revealed spatial and quantitative differences in Wnt-specific transcriptional activation at day 6 in the alcohol group. Days 9 and 14 post-fracture showed that acute alcohol exposure decreased Wnt transcriptional activation, which correlates with the modulation of total β-catenin protein levels observed at these time points. Acute alcohol exposure resulted in significant impairment of fracture callus tissue formation, perturbation of the key Wnt pathway protein β-catenin, and disruption of normal Wnt-mediated transcription. These data suggest that the canonical Wnt pathway is a target for alcohol in bone and may partially explain why impaired fracture healing is observed in alcohol-abusing individuals.

A preliminary histological investigation of gall induction in an unconventional galling system

In an unusual case involving a candidate biological control agent, the histologically complex stem galls of the weevil, Rhinusa pilosa (Coleoptera: Curculionidae) on yellow toadflax (Linaria vulgaris), are rapidly induced during oviposition and reach full size by larval hatch. To investigate gall induction, the oviposition behavior of R. pilosa was described. We experimentally disrupted ovipositing weevils at three key points in the oviposition sequence and compared host-plant tissue responses post disruption, to what occurs during normal gall induction using histological methods. De novo xylem production, intercellular spaces in the cortex, and hyperplasia and hypertrophy of the procambium and pith parenchyma surrounding the egg were some of the tissue- and cellular-level modifications observed only 3–5 days after normal oviposition. Normal gall development was not observed after any of the oviposition disruption treatments, although some of the cellular and tissue responses resembled those found after undisrupted oviposition. Feeding by the female during oviposition canal formation induced wound meristem and callus tissue formation, but no other modifications consistent with gall formation. When females were disrupted about 20 s into oviposition, a homogenously dense substance was observed, which was suspected to be ovipositional fluid. There was minor stem swelling 10 days later and histologically, periclinal cell divisions, de novo xylem, and pith cells with numerous stained plastids were observed as in normal gall development, thus suggesting that ovipositional fluid plays a role in gall induction.

Molecular characterization of a novel AP2 transcription factor ThWIND1-L from Thellungiella halophila

Several AP2/ERF transcription factors are wound-induced regulators, which play key roles in controlling cell dedifferentiation. In this study, a novel AP2/ERF transcription factor, the WIND1-like gene (ThWIND1-L), was isolated and characterized from Thellungiella halophila. ThWIND1-L cDNA clone contained a full open reading frame (ORF) of 1,068 bp encoding 356 amino acids. The predicted amino acid sequence of ThWIND1-L cDNA shared remarkably high degree of identity with the Arabidopsis RAP2.4 (WIND1). The expression of ThWIND1-L rapidly responded to the wound signals, ectopic expression of ThWIND1-L in Arabidopsis plants promoted callus tissue formation without the presence of exogenous hormones. Furthermore, overexpression of ThWIND1-L influenced some primary transcription factors (TFs), which have been shown to be involved in cell dedifferentiation and shoot regeneration. These results indicated ThWIND1-L might be a wound-induced regulator, which played an important role in the control of cell dedifferentiation.

Strontium Is Incorporated into the Fracture Callus but Does Not Influence the Mechanical Strength of Healing Rat Fractures

Strontium ranelate (SrR) is a new agent used in the treatment of osteoporosis and is suggested to reduce bone resorption and increase bone formation. We investigated whether SrR influences the macro- and nanomechnical properties of healing fractures in rats. A closed tibia fracture model was used to study fracture healing in rats after 3 and 8weeks of healing. Two groups of rats were treated with SrR (900mg/kg/day) mixed into the food, while two groups served as control animals. The healing fractures were investigated by three-point bending, dual energy X-ray absorptiometry, energy-dispersive X-ray spectroscopy (EDX), and nanoindentation. There was a 100-fold increase (P<0.001) in serum Sr after 3 and 8weeks of SrR treatment. The callus volume was significantly higher in the SrR-treated group than in control animals (P<0.01) after 3weeks of healing. This was accompanied by a significant increase in callus bone mineral content (P<0.05). However, after 8weeks of healing, no difference was found in either callus volume or bone mineral content. SrR did not influence maximum load or stiffness of the fractures after either 3 or 8weeks of healing. EDX showed that Sr was incorporated into the callus; however, this did not influence the nanomechanical properties. In conclusion, SrR stimulates callus formation but has no effect on callus remodeling. Sr is incorporated into the newly formed callus tissue, but this has no deteriorating effect on the mechanical properties of rat tibial fractures at either the macroscopic or nanoscopic level after 3 or 8weeks of healing.

Correlations between strength and quantitative computed tomography measurement of callus mineralization in experimental tibial fractures

Background The evaluation of fracture healing in the clinic has not changed significantly during the past few decades, despite the development of modern tissue-imaging tools. Recent publications have reported significant and interesting associations between biomechanical properties and quantitative computed tomography data of fractures and grafts. We therefore studied the correlations between the strength and segmented quantitative computed tomography data of tibial diaphyseal fractures. Methods Forty male rats received a tibial-shaft osteotomy that was initially stabilized with either intramedullary nailing or external fixation. Evaluation at 30 and 60 days post-osteotomy included X-ray, quantitative computed tomography and bending testing. Quantitative computed tomography data were segmented by voxel density into soft callus (171–539 mg/cm 3), hard callus (540–1199 mg/cm 3) and cortical bone (≥ 1200 mg/cm 3), and volumetric bone mineral density was calculated. Findings All fractures demonstrated pronounced formation of soft and hard callus tissues at 30 days post-osteotomy, and at 60 days the cortical bone volume was significantly increased with callus resorption. Bending strength correlated significantly and positively with fracture-site cortical bone volume and volumetric bone mineral density in the intramedullary nailed group in the early phase of healing. Interpretation Quantitative computed tomography was used to quantify characteristic secondary healing. The observed correlations indicate that biomechanically important mineralization can be measured by quantitative computed tomography in the early phase of healing in flexibly fixed fractures.

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 muM naphthalene acetic acid and 2.22 muM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.

Ectopic expression of ARR1ΔDDK in tobacco: alteration of cell fate in root tip region and shoot organogenesis in cultured segments

A specific deleted version of ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) lacking the signal receiver domain (1–152 amino acids)-coding sequence, referred to as ARR1ΔDDK, was amplified using Arabidopsis thaliana cDNA prepared from adult leaves and transferred into the genome of Nicotiana tabacum cv. Samsun under the transcriptional control of a β-estradiol-inducible expression system. The ectopic expression of ARR1ΔDDK affected the morphology of transgenic seedlings and their segments in vitro. In the presence of an inducer, β-estradiol, ectopic expression of ARR1ΔDDK induced only the formation of soft, pseudo-bulbous tissue in the root tip region of intact seedlings, which appeared similar to callus generated on a hypocotyl segment in the presence of 2,4-d and 6-benzyladenine (BA), both at 1 μM. Those callus tissues on the root tip region could not generate shoots unless 1 μM BA was supplied. In segment culture, ectopic expression of ARR1ΔDDK induced callus-like tissue around the cut-end of cotyledon and hypocotyl segments with occasional shoot formation, suggesting that the expression of ARR1ΔDDK could substitute for the effects of cytokinin on these segments. Additionally, treatment with only β-estradiol induced NtWUS, a WUS ortholog in tobacco, which was detected during the process of callus tissue formation in the root tip region and also in cotyledon or hypocotyl segments. These findings suggest that the NtWUS might be associated in the transdifferentiation process caused by the functional regulation of ARR1ΔDDK in transgenic tobacco seedlings.

Formation and spread of callus tissue and tangential rows of resin ducts in Larix decidua and Picea abies following rockfall impacts

After mechanical wounding, callus tissue and tangential rows of traumatic resin ducts (TRDs) are formed in many conifer species. This reaction can be used to date past events of geomorphic processes such as rockfall, debris flow and snow avalanches. However, only few points are known about the tangential spread or the timing of callus tissue and TRD formation after wounding. We analyzed 19 Larix decidua Mill. (European larch) and eight Picea abies (L.) Karst. (Norway spruce) trees that were severely damaged by rockfall activity, resulting in a total of 111 injuries. Callus tissue appeared sparsely on the cross sections and was detected on only 4.2% of the L. decidua samples and 3.6% of the P. abies samples. In contrast, TRDs were present on all cross sections following wounding and were visible on more than one-third (34% in L. decidua and 36.4% in P. abies) of the circumference where the cambium was not destroyed by the rockfall impact. We observe different reactions in the trees depending on the seasonal timing of wounding. The tangential spread of callus tissue and TRDs was more important if the injury occurred during the growth period than during the dormant season, with the difference between seasons being more pronounced for callus tissue formation than for TRD formation. We observed an intra-annual radial migration of TRDs with increasing tangential distance from the wound in 73.2% of the L. decidua samples and 96.6% of the P. abies samples. The persistence of TRD formation in the years following wounding showed that only L. decidua trees produced TRDs 2 years after wounding (10.5%), whereas P. abies trees produced TRDs 5 years after wounding (> 50%).

Rockfall and snow avalanche impacts leave different anatomical signatures in tree rings of juvenile Larix decidua

Rockfall and snow avalanche events often cause injury to European larch (Larix decidua Mill.) trees, giving rise to the formation of callus tissue and tangential rows of traumatic resin ducts (TRDs). We analyzed and quantified anatomical reactions of juvenile trees injured before the start of the growing season by snow avalanches (15 trees, 324 cross sections) or rockfalls (18 trees, 270 cross sections). Traumatic resin ducts were observed in the growth ring formed following injury in 94.3% of the rockfall samples and 87.3% of the snow avalanche samples. Traumatic resin ducts were formed at the beginning of the new annual ring around wounds caused by rockfalls. In contrast, in trees injured by snow avalanches, TRDs were not formed until after the formation of several rows of early earlywood (EE) tracheids (mean +/- SD = 4.19 +/- 2.56 rows). The dimensions of the EE tracheids observed in the snow avalanche samples were greatly reduced in the tissues bordering the wound, with radial width reaching an average of only 50% and lumen cross-sectional area an average of only 46% of pre-event values. It is therefore possible to differentiate injuries due to past snow avalanches from injuries due to rockfall based on anatomical growth reactions in the tissues bordering scars.

Prediction of biomechanical stability after callus distraction by high resolution scanning acoustic microscopy

Accurate clinical prediction of the resistance to fracture after callus distraction requires a detailed understanding of structural and elastic properties of the newly formed bone. We investigated 26 sheep that underwent middiaphyseal callus distraction at a rate of 0.5 mm every 12 h for 30 d using a standard unilateral fixator system. The sample population included four groups undergoing different treatments to improve bone healing, including bone grafting and the local application of growth factors. All animals were sacrificed eight weeks after the end of distraction. The fracture forces of the lengthened tibia and the contralateral control tibia from each animal were evaluated by biomechanical (four-point bending) testing. The microstructure and anisotropic acoustic impedance distributions were assessed by quantitative 50-MHz scanning acoustic microscopy. The relationships between resistance to fracture, structural properties and acoustic impedance of the newly formed callus tissue and adjacent cortical tissue were investigated. A significant linear multivariate regression model was developed that predicts the fracture force with a high accuracy (RMSE = 248 N, R 2 = 0.86, p < 0.0001). (E-mail: kay.raum@medizin.uni-halle.de)

Somatic embryogenesis of Gentiana cruciata (L.): Histological and ultrastructural changes in seedling hypocotyl explant

Structure and ultrastructure changes that occurred during tissue culture of upper explants of hypocotyl (adjacent to cotyledons) of 10-d-old seedlings of Gentiana cruciata were studied. The explants were cultured on Murashige and Skoog induction medium supplemented with 1.0 mg l−1 dicamba +0.1 mg l−1 naphthaleneacetic acid +2.0 mg l−1 benzyladenine +80.0 mg l−1 adenine sulfate. The initial response of the explant and callus formation were ultrastructurally analyzed during the first 11 d of culture. After 6–8 wk, various methods were employed to collect evidence of indirect somatic embryogenesis. After 48 h of culture, the earliest cell response was cell division of epidermis and primary cortex. There were numerous disturbances of karyo- and cytokinesis, leading to formation of multinuclear cells. With time, the divisions ceased, and cortex cells underwent strong expansion, vacuolization and degradation. About the 6th day of culture, callus tissue proliferated and the initial divisions of vascular cylinder cells were observed. Their division appeared normal. Cells originating from that tissue were small, weakly vacuolated, with dense cytoplasm containing active-looking cell organelles. Numerous divisions occurred in the vascular cylinder, which led to its expansion and the formation of embryogenic callus tissue. During the 6–8th wk of culture, in the proximal end of the explant, masses of somatic embryos were formed from outer parts of intensively proliferating tissue.

Principles of callus distraction

Callus distraction is based on the principle of regenerating bone by continuous distraction of proliferating callus tissue. It has become the standard treatment of significant leg shortening and large bone defects. Due to many problems and complications, exact preoperative planning, operative technique and careful postoperative follow-up are essential. External fixators can be used for all indications of callus distraction. However, due to pin tract infections, pain and loss of mobility caused by soft tissue transfixation, fixators are applied in patients with open growth plates, simultaneous lengthening with continuous deformity corrections, and increased risk of infection. Distraction over an intramedullary nail allows removal of the external fixator at the end of distraction before callus consolidation (monorail method). The intramedullary nail protects newly formed callus tissue and reduces the risk of axial deviation and refractures. Recently developed, fully intramedullary lengthening devices eliminate fixator-associated complications and accelerate return to normal daily activities. This review describes principles of callus distraction, potential complications and their management.

Identification of Germinisoforms in Wheat Callus

Application of auxin causes cells of the wheat embryo to undergo dedifferentiation and form callus tissue. The synthesis of germin isoforms (G, G’ and ωG) has been selectively associated with different phases of embryonic development and callus formation. G and G’ germin isoforms are found to be associated with germination. ωG is an embryogenesis related germin isoform. It is clearly important to distinguish whether callus induction caused a precocious initiation of embryonic maturation-specific pseudogermin synthesis, or whether it effected a switch in the pattern of protein synthesis, eliciting accumulation of the normally germination-related germin proteins. To distinguish between these alternatives, it is necessary to identify the germin protein isoforms accumulated during callus formation. Following the electrophoretic separation of callus protein extracts, the position of germin isomers were detected by Western-blotting. The immunoreactive polypeptides accumulating in callus tissue were determined to be exclusively germin, rather than ωG.

Cell differentiation, secondary cell-wall formation and transformation of callus tissue of Pinus radiata D. Don.

Tracheid and sclereid differentiation was induced in callus cultures of Pinus radiata D. Don by culturing on a basal medium containing activated charcoal but no phytohormones; sclereids differentiated in callus derived from xylem strips, but not in callus derived from hypocotyl segments. The tracheids differentiated in hypocotyl-derived callus had helical, scalariform, reticulated or pitted secondary cell-wall patterns, but those differentiated in xylem-derived callus had a reticulate or pitted pattern. The thickened tracheid and sclereid walls contained lignin as indicated by the red colour reaction given with phloroglucinol-HCl. The presence of lignin in the cell walls of differentiated callus was confirmed using pyrolysis gas chromatography-mass spectrometry by the detection of phenylpropanoid components derived from lignin. Lignin was also detected using solid-state (13)C cross-polarisation/magic-angle spinning nuclear magnetic resonance spectroscopy and quantified as thioglycolic acid lignin. Monosaccharide analyses of the cell walls isolated from differentiated and undifferentiated calli showed that the cell walls of the differentiated calli contained higher proportions of glucose and mannose, consistent with the presence of greater proportions of gluco- and/or galactogluco-mannans in the secondary cell walls of the differentiated cells. A protocol for the stable transformation of undifferentiated, xylem-derived cultures was successfully developed. Transgenic cell lines were established following Biolistic particle bombardment with a plasmid containing the coding region of the nptII gene and the coding region of the cad gene from P. radiata. Expression of the nptII gene in transgenic lines was confirmed by an NPTII-enzyme-linked immunosorbent assay. The overexpression of cad in the transgenic lines resulted in a down-regulation of cinnamyl alcohol dehydrogenase (EC 1.1.1.195) expression.

The initial phase of fracture healing is specifically sensitive to mechanical conditions

Interfragmentary movements affect the quality and quantity of callus formation. The mounting plane of monolateral external fixators may give direction to those movements. Therefore, the aim of this study was to determine the influence of the fixator mounting plane on the process of fracture healing. Identically configured fixators were mounted either medially or anteromedially on the tibiae of sheep. Interfragmentary movements and ground reaction forces were evaluated in vivo during a nine week period. Histomorphological and biomechanical parameters described the bone healing processes. Changing only the mounting plane led to a modification of interfragmentary movements in the initial healing phase. The difference in interfragmentary movements between the groups was only significant during the first post-operative period. However, these initial differences in mechanical conditions influenced callus tissue formation significantly. The group with the anteromedially mounted fixator, initially showing significantly more interfragmentary movements, ended up with a significantly smaller callus diameter and a significantly higher callus stiffness as a result of advanced fracture healing. This demonstrates that the initial phase of healing is sensitive to mechanical conditions and influences the course of healing. Therefore, initial mechanical stability of an osteosynthesis should be considered an important factor in clinical fracture treatment.

A novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants

A direct somatic embryogenesis and secondary embryogenesis protocol was developed for seven cereal species, thus providing a new vista for in vitro plant genetic transformation or propagation. This paper describes a novel process that has been successfully developed for efficient regeneration of a wide range of cereal species and genotypes. This tissue culture and regeneration system does not require formation of callus tissues and takes approximately 2 months to complete, shorter than any of the currently available systems requiring 3-4 months. Rapid induction of direct somatic embryogenesis in barley (Hordeum vulgare), common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu), rye (Secale cereale) and oats (Avena sativa) was induced from excised immature scutellum on DSEM medium. Newly developed globular embryos were cultured on SEM medium for a second cycle of embryogenesis followed by germination (GEM medium) and regeneration of embryos into normally growing green and fertile plants. In vitro techniques to induce direct somatic embryogenesis, secondary embryogenesis and plant regeneration from these cereals require a specific sequence of defined media and controlled environments. The sequence and the timing of the media used, as well as their hormonal composition and balance are critical aspects of this process. The organic and mineral compositions of these media are not new but are important for supporting and sustaining rapid growth of the tissues.

Developmental stages and fine structure of surface callus formed after debarking of living lime trees (Tilia sp.).

Wounding of trees by debarking during the vegetative period sometimes results in the formation of callus tissue which develops over the entire wound surface or on parts of it. This light and transmission electron microscopy study of living lime trees found that the formation of such a surface callus is subdivided into three stages. During the first stage, numerous cell divisions take place in regions where differentiating xylem remains at the wound surface after debarking. This young callus tissue consists of isodiametric parenchymatous cells. Cambium cells, sometimes also remaining at the wound surface, collapse and do not contribute to callus formation. During the second stage, cells in the callus undergo differentiation by forming a wound periderm with phellem, phellogen and phelloderm. In the third stage, a cambial zone develops between the wound periderm and the xylem tissue laid down prior to wounding. This process is initiated by anticlinal and periclinal divisions of a few callus cells only. Later this process extends tangentially to form a continuous belt of wound cambium. Subsequently, this cambium produces both wound xylem and wound phloem and thus contributes to further thickening.

Response to auxin changes during maturation-related loss of adventitious rooting competence in loblolly pine (Pinus taeda) stem cuttings.

Hypocotyl cuttings (from 20- and 50-day-old Pinus taeda L. seedlings) rooted readily within 30 days in response to exogenous auxin, while epicotyl cuttings (from 50-day-old seedlings) rarely formed roots within 60 days. Responses to auxin during adventitious rooting included the induction of cell reorganization and cell division, followed by the organization of the root meristem. Explants from the bases of both epicotyl and hypocotyl cuttings readily formed callus tissue in response to a variety of auxins, but did not organize root meristems. Auxin-induced cell division was observed in the cambial region within 4 days, and later spread to the outer cortex at the same rate in both tissues. Cells at locations that would normally form roots in foliated hypocotyl cuttings did not produce callus any differently than those in other parts of the cortex. Therefore, auxin-induced root meristem organization appeared to occur independently of auxin-induced cell reorganization/division. The observation that N-(1-naphthyl)phthalamic acid (NPA) promoted cellular reorganization and callus formation but delayed rooting implies the existence of an auxin signal transduction pathway that is specific to root meristem organization. Attempts to induce root formation in callus or explants without foliage were unsuccessful. Both the cotyledon and epicotyl foliage provided a light-dependent product other than auxin that promoted root meristem formation in hypocotyl cuttings.

Hydraulic contribution in cell elongation of tissue‐cultured plants: growth retardation induced by osmotic and temperature stresses and addition of 2,4‐dichlorophenoxyacetic acid and benzylaminopurine

ABSTRACTThis work was undertaken to determine the growth parameters of Lockhart’s equation for finding which component was predominantly contributing to the cell expansion rates of plants subjected to environmental stresses under tissue‐culture conditions. Embryos isolated from soybean (Glycine max [L.] Merr.) and kidney bean (Phaseolus vulgaris L.) seeds were grown under tissue‐culture conditions. The water potential of culture media ranged from − 0·02 to − 0·94 MPa so that nutrient deficiency and salt stress conditions could be applied. Additionally, the temperature of culture conditions was set from 10 to 40 °C to apply low‐temperature and high‐temperature stresses on plants grown at the optimum concentration of culture medium. Cell expansion could be inhibited completely by adding 2,4‐dichlorophenoxyacetic acid and benzylaminopurine to culture media to form callus tissue. The sizes of the water potential gradient between the water source and elongating cells correlated with the speed of growth rates under nutrient deficiency, salt stress, growth retardation induced by plant hormones, low‐temperature and high‐temperature conditions in the present study, indicating that cell expansion rates were mainly associated with how much water could be absorbed by elongating cells regardless of the kinds of environmental stress conditions applied.