Callus Type Research Articles

Callus formation and plant regeneration in various Lilium species and cultivars

In researching the application of genetic transformation to lily breeding, callus formation from cultured explants and plant regeneration from induced calluses were examined in 33 Lilium genotypes, 21 species, three Asiatic hybrids, two LA hybrids, two Longiflorum hybrids, three Oriental hybrids, and two Trumpet hybrids. Seed, bulb scale, leaf, or filament explants were placed on a medium containing 4.1 μM 4-amino-3,5,6-trichloropicolinic acid (picloram; PIC) and cultured in the dark. After 2 mo., callus formation was observed in 30 genotypes, and a formation frequency of more than 50% was obtained in 24 genotypes. Bulb scale and filament explants showed great ability to form calluses, whereas seeds had poor ability. Most of the induced calluses were yellow and had a nodular appearance. When subcultured onto the same fresh medium, twofold or more increases in callus mass were obtained in 1 mo. for 15 genotypes. Callus lines showing sustained growth 1 yr after the initiation of subculture were examined for their ability to produce shoots on a medium without plant growth regulators (PGRs) and a medium containing 22 μM 6-benzyladenine (BA). Shoot regeneration was observed in all genotypes examined, and a regeneration frequency of over 80% was obtained in 20 genotypes. Initial explants used for callus induction and callus type (nodular or friable) had no effect on shoot regeneration. Most of the regenerated shoots developed into complete plantlets following their transfer to a PGR-free medium.

ADVENTITIOUS SHOOT FORMATION ON TEAK (TECTONA GRANDIS L.F.) CALLUS CULTURES DERIVED FROM INTERNODAL SEGMENTS

ADVENTITIOUS SHOOT FORMATION ON TEAK (TECTONA GRANDIS L.F.) CALLUS CULTURES DERIVED FROM INTERNODAL SEGMENTS

Produção de frutanos em calos e plântulas clonadas in vitro de Viguiera discolor Baker (Asteraceae)

Viguiera discolor Baker é uma espécie nativa do cerrado, cuja sobrevivência está ameaçada pela invasão de espécies exóticas. Considerando sua elevada produção e a vasta aplicação de frutanos, o presente trabalho foi conduzido visando à obtenção in vitro dessa espécie e à prospecção desses carboidratos nessas condições. Sementes foram germinadas in vitro, em meio MS modificado, e após cinco semanas de incubação, nós caulinares foram isolados e incubados no mesmo meio adicionado de 0,5 mg L-1 de ANA, regenerando plantas uniformes, raízesnão espessadas, raízes tuberosas e estruturas semelhantes a calo (calo tipo1), formadas na região caulinar basal. Análise desse material evidenciou a presença de frutanos do tipo inulina nas raízes tuberosas e nos calos tipo 1. Na presença de 2,4-D obteve-se a formação de calos friáveis (calo tipo 2), nos quais também foram detectados frutanos e suas enzimas de síntese sacarose: sacarose 1-frutosiltransferase (SST) e frutano: frutano 1-frutosiltransferase (FFT). Embora em concentrações menores às observadas nas plantas cultivadas sob condições naturais, o material produzido in vitro apresentou frutanos do mesmo tipo e razão SST/FFT menor do que um. Em meio de cultura sem hormônios, foi verificada a regeneração de 50% de plantas a partir dos nós caulinares. A propagação de V. discolor in vitro pode viabilizar a multiplicação e a preservação da espécie, bem como a produção de frutanos nessas condições.

Long-lasting Capsicum baccatum ‘Organogenic callus’ formation

Capsicum regeneration is often obtained through direct (adventious) regeneration and only a few reports claim indirect regeneration (through callus) as an option to obtain complete plants. A possible reason for this may be because Capsicum cultures offer a narrow window in time for the regeneration process to occur, and after that, the ability to regenerate plants rapidly diminishes. In this study, the C. baccatum radicle-side half-seed was the explant of choice to induce the formation of callus on semisolid Murashige and Skoog (1962) medium supplemented with 5 mgl−1 (22.2μM) 6-benzylaminopurine, 1 mgl−1 (5.7μM) indole-3-acetic acid, and 2 mgl−1 (6.1μM) gibberellic acid. Organogenic calluses developed within 4–5 mo. and were subcultured every 2 mo. thereafter. Eventual bud elongation occurred and these shoots developed into complete plants after transfer to medium without plant growth regulators. The organogenic callus type retained its organogenic ability for more than 3 yr.

Sélection et évaluation de lignées de cals stables et tolérantes vis-à-vis du stress salin chez le citrange ‘Troyer’ [Citrus sinensis(L.) ×Poncirus trifoliata(L.) Raf.

Selection and evaluation of stable salt-tolerant callus cell lines of the Troyer citrange. Abstract -- Introduction. Citrus fruits are sensitive to salinity. Our study aimed at selecting cellular lines tolerant to NaCl of citrange Troyer and assessing the characters involved in salt stress responses in callus cultures. Materials and methods. Calli were cultivated on a culture medium containing NaCl whose concentration was gradually increased until it reached 8 g NaCl·L -1 . They were then transferred onto a medium without NaCl to test their independence with respect to the salt, then placed in the presence of 8 g NaCl·L -1 to test their stability. Dosages of the content in soluble Na + , K + , proline and sugars contained in the calli were carried out at the end of the experiment. Results and discussion. Salinity stopped the calli from growing and caused the majority of the explants (sensitive calli) to become brown. Tolerant calli presented a growth comparable with the growth of the controls. The transfer of these calli onto medium without NaCl followed by their transfer onto saline culture medium showed the independence and the stability of the selected character of tolerance. The content in K + of the tolerant calli was close to that of the control calli, but it was greater than that of the sensitive calli. On the other hand, the content in Na + ions in the tolerant and sensitive calli was relatively higher than that of the control calli. Sodium would thus be accumulated in two cellular levels according to the type of callus: invasion of the cytosol (toxic effect) for the sensitive calli or vacuolar partitioning for the tolerant calli. Salinity caused the accumulation of proline and soluble sugars in the tolerant calli but not in the sensitive calli. Conclusions. Stable and tolerant cellular lines in NaCl were obtained starting from embryos of citrange Troyer. This tolerance could be related to changes in the properties of ion migration and in the regulation capacity and ionic partitioning. Accumulation of proline and soluble sugars constitutes a metabolic character to adaptive value that could be an indicator of tol- erance to salinity. Morocco / Citrus / citranges / in vitro culture / callogenesis / salt tolerance

A new selection method for pepper transformation: callus-mediated shoot formation

We used two genes, TMV-CP and PPI1 (pepper-PMMV interaction 1 transcription factor), to transform commercially important chili pepper (Capsicum annuum) inbred lines (P915, P409) by means of Agrobacterium co-culture. Eighteen independently transformed T0 plants were obtained. The most critical point in the pepper transformation protocol was the selection of shoots growing on calli--referred to as callus-mediated shoot formation (indirect shooting)--because shoots not grown from the callus (direct shooting from the wounded surface) developed into non-transformants. Selection of the correct right callus type also proved to be an important requirement for obtaining transformed peppers. Six different types of callus developed during the selection process. Shoots regenerated from two of these types, while one type regenerated significantly more shoots than the other types, suggesting that the capacity for shoot formation is callus type-specific. Although the transformation rate was low, transformation via callus-mediated shoot formation proved to be reproducible and was confirmed by Southern and Northern blot analyses. Based on the experimental data, we have succeeded in developing a new protocol for the selection and transformation of pepper and expect that it will be used in the future for pepper transformation.

Efficient plant regeneration in Holarrhena antidysenterica Wall., from shoot segment-derived callus

A procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica, through shoot segment-derived callus. Explants used for callus induction were shoot segments derived from 14-d-old axenic plants on Murashige and Skoog (MS) medium supplemented with 15 μM N6-benzyladenine (BA). A white friable type of callus was obtained in 4.52 μM 2,4-dichlorophenoxyacetic acid and 2.32 μM kinetin which did not have the potentiality to regenerate. High-frequency shoot differentiation was achieved on transferring the friable callus to MS medium supplemented with 17.8 μM BA and 8.0 μM naphthaleneacetic acid. The highest percentage of calluses forming shoots (65.06±2.26) was achieved in this medium. The organogenetic potential of the regenerating callus was influenced by the age of the culture. Rooting was achieved on the shoots using MS medium with 25 μM indolebutyric acid. The plantlets were acclimatized and established in soil. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the donor plants.

Effect of genotype on callus induction, shoot regeneration, and phenotypic stability of regenerated plants in the greenhouse of Primula ssp.

Genotypic differences between six genotypes of Primula vulgaris could be observed in callus induction rate, type of callus, root formation during the callus phase, and shoot regeneration rate. The shoot regeneration rate ranged from zero to 11.6 shoots per explant. There was no correlation between callus induction rate and shoot regeneration rate. Callus consistency and colour were an indicator of the organogenetic capacity of callus. An experiment with different periods of treatment with 4.0 mg l 2,4-dichlorophenoxyacetic acid and 2.0 mg l21 thidiazuron revealed that the shoot regeneration rate varied tremendously between genotypes. In two genotypes a period of 8 weeks on medium with plant growth regulators was sufficient to induce shoot regeneration. In three other genotypes a longer induction period was not able to overcome low regeneration capacity. However an increase in shoot regeneration rate was observed after 16iV32 weeks of induction. Phenotypic stability was also strongly dependent on genotype. In three genotypes the majority of regenerated plants looked normal and were diploid. Aberrations like abnormal growth habit, crinkly leaves, deviation of flower colour or lack of pollen formation occurred in only one genotype at a very low frequency (1.5 genotypes between 12.5 and 18.1 regenerants was tetraploid.

Insecticidal activity of a cryia(c) transgene in callus derived from regeneration-recalcitrant cotton (Gossypium hirsutum L.)

The insecticidal effectiveness of a δ-endotoxin Cry protein from Bacillus thuringiensis in non-regenerable callus of a commercial Gossypium hirsutum L. variety was investigated. Two transgenic callus types were generated. The first callus type harbored the cry1A(c) gene and the hygromycin B phosphotransferase hpt selectable marker gene. The second callus type, the transgenic control, carried the marker genes β-glucuronidase (GUS) and hpt. Growth and survival rates of three major cotton moth species, Pectinophora gossypiella, Helicoverpa armigera, and Spodoptera littoralis, were examined with aseptic neonates reared on callus. Normal larval development occurred in all species supplied with non-transgenic callus, but insects died, or their growth was severely restricted, when reared on transgenic callus harvested from hygromycin B-supplemented medium. Development of larvae on transgenic control and on non-transgenic callus became very much alike after the transgenic control tissue had been subcultured on a hygromyein B-free medium for about 100 d prior to the insect-callus bioassay. Accordingly, for detection of Bt toxin activity without the interference of the influence of hygromycin B on insects, cry1A(c) callus was infested with insects after it had been propagated for more than 100 d on a medium free of the antibiotic. Under these experimental conditions all P. gossypiella and H. armigera, and most S. littoralis neonates died, and the growth (e.g., weight increment) of S. littoralis survivors was markedly impeded by cry1A(c) callus. Three new findings emerge from this study: first, P. gossypiella, a pest feeding in the field on bolls only, can be grown in vitro on cotton callus; second, in a host which is recalcitrant in terms of plant regeneration, the biological potency of an insectdetrimental transgene can nevertheless be evaluated by generating a transgenic host callus and conducting in vitro transgenic callus-insect assays; and third, our results suggest that hygromycin B is toxic to lepidopteran larvae.

Somatic Embryos Produced from Aseptic Seedlings of Wild Cyclamen Species.

Somatic embryos were produced using aseptic seedlings of wild Cyclamen species. The seeds of 18 wild Cyoclamen species were cultured in the dark at 15-20°C until germination, and then maintained under a 16/8 light/dark photoperiod (20-30μmol m-2 s-1 provided by fluorescent lamps) at 24°C. Modified MS medium supplemented with 30.0gl-1 sucrose and 3.0gl-1 gellan gum was used for culture of aseptic seedlings. The seeds of C. balearicum, C. creticum, C. intaminatum and C. repandum did not germinate. Aseptic seedlings were obtained from 14 wild Cyclamen species : C. africanum, C. cilicium, C. coum, C. cyprium, C. graecum, C. hederifolium var. album, C. libanoticum, C. mirabile, C. parviflorum, C. persicum, C. pseudibericum, C. purpurascens, C. rohlfsianum and C. trochopteranthum. Sectioned petioles and leaves from the seedlings were inoculated on two types of induction media. The induction was carried out in the dark at 24°C. Among the 14 species, only C. cyprium, C. libanoticum, and C. parviflorum did not produce calli. The calli were subcultured and transferred to a somatic induction mealum without plant growth regulators. Variations in the type of callus, the frequency of callus formation and the potential for somatic embryogenesis were observed among the species. Somatic embryogenesis was achieved in C. africanum, C. cilicium, C. persicum, C. rohlfsianum and C. trochopteranthum and shoot formation in C. africanum and C. hederifolium var. album for the first time in this experiment.

Influence of genotype and culture medium on callus formation and plant regeneration from immature embryos of Triticum turgidum Desf. cultivars

AbstractTwelve durum wheat cultivars were evaluated for their response to in vitro tissue culture. Zygotic immature embryos were used to induce callus formation using four different Murashige and Skoog‐based media. Each contained 9.05 μM 2,4‐dichlorophenoxy acetic acid but differed in their carbon source (sucrose or maltose) and the presence of NaCl (0 mM or 40 mM). The influence of both genotype and medium on the type and percentage of callus produced was observed. Calli were either compact and frequently embryogenic, or soft and watery. Percentages ranged from 54 to 100%, depending upon genotype and induction medium. All calli were then plated on a regeneration medium containing 20 g/l sucrose, 2.68 μM 1‐naphthaleneacetic acid and 2.22 μ 6‐benzylaminopurine. The regeneration of plantlets was higher from compact than from soft calli, with a strong dependence on genotype and type of induction medium used. MSm induction medium (30 g/l maltose) and MS40s (30 g/l sucrose plus 40 mM NaCl) were best for inducing compact calli, and gave the highest proportion of regenerated plants. The in vitro response (number of total shoots from a compact callus/number of embryos plated) was higher for immature embryos of ‘Baztan’, ‘Bradano’ and ‘Don Pedro’. These cultivars are a good starting material for experiments involving transformation of calli from zygotic immature embryos.

Multiple Micronutrient Deficiencies in a Child Fed an Elemental Formula

Multiple Micronutrient Deficiencies in a Child Fed an Elemental Formula

Rice and Phragmites: effects of organic acids on growth, root permeability, and radial oxygen loss to the rhizosphere

Young Phragmites plants were grown in two cocktails of monocarboxylic acids (C(1)-C(5)) at pH 6, where the concentration of each acid was innocuous and the total undissociated (potentially toxic) concentrations were 0.35 mmol/L and 0.42 mmol/L. Rice plants were subjected to 1.5 mmol/L acetic acid at pH 4.5 (undissociated concentration = 1.05 mmol/L). In Phragmites, each cocktail curtailed root growth especially and induced premature shoot senescence. In both species, after 3-5 d of treatment, radial oxygen loss (ROL) from apical regions of adventitious roots, and from Phragmites laterals, was reduced to very low values and associated with cell wall lignification and suberization in the surface cell layers. At later stages of treatment, rice responded to acetic acid in similar ways to Phragmites, with the development of intercellular and callus type occlusions in the gas space system, vascular blockages, and the failure of laterals to emerge. The results are relevant to the supply of oxygen from Phragmites roots to sediments for the phytopurification of waste waters, to the efflux of methane and carbon dioxide from wetlands, and to rice cultivation.

Endogenous hormone concentrations and embryogenic callus development in wheat

Immature zygotic embryos of two wheat (Triticum aestivum L.) genotypes, known for their different ability to generate embryogenic callus, were used as initial explants to establish callus cultures. Embryogenic and non-embryogenic calluses were obtained from the competent genotype (`Combi'), while only non-embryogenic callus was produced by the incompetent one (`Devon'). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. The endogenous hormone concentrations (free indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins 1, 3 and 20 [GAs], zeatin/zeatin riboside [Z/ZR] and N6[Δ2-isopentenyl] adenine/ N6[Δ2-isopentenyl] adenosine; [iP/iPA]) of the initial explants were determined by means of radio-immunoassay and showed that the only difference was the higher concentration of ABA found in the embryos of the most competent genotype; whose embryos showed a reduced rate of precocious germination. When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses. Higher concentrations of endogenous free IAA were typical of embryogenic callus cultures. It was also observed that a loss in the embryogenic competence of the calluses, due to a prolonged time of culture, occurred concomitantly with a reduction in free IAA concentrations, practically to the concentrations found in the non-embryogenic calluses.

Hormonal status of maize initial explants and of the embryogenic and non-embryogenic callus cultures derived from them as related to morphogenesis in vitro

Endogenous hormone levels (indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins 1, 3 and 20 [GAs], zeatin/zeatin riboside [Z/ZR] and N 6[Δ 2-isopentenyl] adenine/ N 6[Δ 2-isopentenyl] adenosine; [iP/iPA]) were analysed in immature maize zygotic embryos of two maize ( Zea mays L.) genotypes, known for their distinct ability to generate embryogenic (E) callus. No differences were found among genotypes in the hormone contents of the embryos. These embryos were also used as initial explants to establish callus cultures. E and non-embryogenic (NE) calli were obtained from the competent genotype (A188), while only NE callus was produced by the incompetent one (B73). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. When analysing the endogenous hormone levels in the various callus types generated in each genotype, it was found that only differences in the IAA levels accounted for variations in the morphogenic properties of the calli. Higher levels of endogenous IAA were typical of embryogenic callus cultures. It was also observed, that a loss in the embryogenic competence of the calli, due to a prolonged time of culture, occurred concomitantly with a reduction in the IAA levels, practically to the levels found in the non-embryogenic calli.

Somatic embryogenesis in Asparagus officinalis can be an in vitro selection process leading to habituated and 2,4-D dependent embryogenic lines

Long-term embryogenic lines were repeatedly obtained from nine asparagus ( Asparagus officinalis L.) genotypes by the selection of rare events, which consisted of the emergence of either a few somatic embryos or an embryogenic callus from a restricted area of a primary callus. In the first case, somatic embryos emerged from 1 % of calli induced with naphtaleneacetic acid and transferred to a medium without auxin. Isolated and subcultured on hormone free medium, these embryos developed habituated embryogenic lines (H lines) growing by adventive embryogenesis. In the second case, 3 % of primary calli developed then subcultured on 2,4-dichlorophenoxyacetic acid (2,4-D) produced a new type of friable and yellowish-white callus, constituted of clusters of globular somatic embryos which can be continuously maintained on 2,4-D (2,4-D lines). Among 2,4-D lines, two types were identified by subculturing them on hormone–free medium. Half of the 2,4-D lines were habituated and half were 2,4-D dependent. Most plants regenerated from H lines exhibited a strong increase in embryogenic capacity compared to control plants, unlike plants regenerated from the 2,4-D dependent lines. This increased embryogenic capacity was transmitted to the progeny as a monogenic dominant trait. H lines would therefore be issued from mutation(s) occurring in vitro, conferring both the embryogenic and habituated phenotypes. On the contrary, in the 2,4-D dependent lines, the embryogenic processes appeared to remain under exogenous auxin control and no evidence of a mutational origin could be inferred from the behaviour of regenerated plants.

Plant regeneration from callus and suspension cultures of Valeriana edulis ssp. procera via simultaneous organogenesis and somatic embryogenesis

Plants of the genus Valeriana produce irioid esters of biotechnological interest, known as valepotriates. The regeneration of Valeriana edulis ssp. procera via indirect organogenesis and somatic embryogenesis from leaf-derived callus and suspension cultures, is presented. For induction of callus, leaves were cultured on semisolid MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin. Embryogenic and organogenic callus development was observed after 16 weeks. Calli were transferred to semisolid and liquid Murashige and Skoog (MS) medium supplemented with kinetin and 1-naphthaleneacetic acid (NAA). Appearance of shoot and somatic embryo occurred 4 weeks later. Shoots were rooted and somatic embryos were germinated and were transferred to the greenhouse. A better organogenic and embryogenic response was observed from suspension cultures. Histological observations of morphogenic callus revealed that both somatic embryos and shoots arose from the same type of callus.

Relationship Between Radiologic Morphology of the Bone Lengthening Formation and Its Complications

The objective was to study the different types of lengthened bone regeneration and their development during the various phases of the process to correlate them with patient factors and the surgical technique used, and to establish a possible relation between the development of the bone lengthening formation and the problems or complications. The authors studied the radiographs of a random group of 55 patients taken at three points during the course of treatment. The callus was classified with regard to its transverse diameter and the presence or absence of hypodense areas. The overall callus type was significantly influenced by the etiology, the osteotomy site, and the percentage lengthened. The percentage by which the limb was lengthened at the beginning of the process influences the overall morphology of the callus. Poor callus had been lengthened the most, atrophic callus the least. There was a correlation between the morphology of the overall callus at the end of treatment and the percentage lengthened, and between the percentage lengthened and the presence of bands at the end of treatment. The authors also found a significant correlation between age and the appearance of bands at the end of distraction. A central band was found among younger patients. The type of osteotomy affected the overall callus at the end of distraction and at the end of treatment and also influenced the transverse diameter. All the elongations with poor bone formation at the end of treatment were found to have undergone a diaphyseal osteotomy. The most common complication at the first follow-up and at the end of distraction was angulation. The diameter of the callus and the presence of bands at the end of treatment were significantly related to the complications. Fracture occurred in the first 2 weeks after removal of the external fixator in 88% of cases and in the third and fourth week in the rest. However, the segment had no significant influence on the appearance of complications. Lengthened callus with incomplete trabecular formations and hypodense areas at the end of the treatment has a high risk of fracture at the end of treatment. Callus with axial deviation, hypodense areas, or an insufficient transverse diameter during the lengthening procedure must be manipulated so that it reaches the maturing phase in better condition.

Plant regeneration from leaf sheath cultures of some rabi sorghum cultivars

High frequency callus induction was obtained from young leaf sheath segments of four rabi type sorghum cultivars viz., M 35-1, SPV-86, A-1 and GRS-1 on Murashige and Skoog (MS) medium supplemented with 2 mgl − 1 of 2,4-dichlorophenoxyacetic acid (2,4-D). The callus was unorganised in the beginning, but became highly organised and nodular within 2–3 months. On MS medium without 2,4-D, plantlets occurred at high frequency through somatic embryogenesis. Addition of 0.5 mgl − 1 of 6-benzyl aminopurine improved the formation of plants Genotypic influence with regard to the frequency of callus Induction, type of callus, pigment production and regeneration of plantlets were observed.

SOMATIC EMBRYOGENESIS IN TULIP

Because the natural propagation rate of tulip is low, modern breeding requires a rapid in vitro propagation method. The best method available at the moment, adventitious shoot formation on stem explants, has a low propagation factor and is laborious, time-consuming and hence expensive. It is the long term aim of the present study to develop a propagation method, consisting of a cell suspension phase, and a regeneration phase, during which the cell clusters regenerate through somatic embryogenesis. A system like this would enable the rapid production of large numbers of bulblets at low costs. In order to obtain friable callus for the start of a cell suspension, bulb scale and stem explants were cultured on a nutrient medium containing the auxins 2,4-D or Picloram at a concentration range from 0.5-50 μM. Undifferentiated and 'meristematic' type of callus on bulb scale explants an were induced. The meristematic type seemed the most suitable to start a liquid culture. In liquid medium the meristematic nodules divided into smaller units, which could be released from the explant by shaking. On stem explants on medium with 5 or 50 μM 2,4-D or Picloram somatic embryos were formed. Morphological and anatomical data on the development of these somatic embryos are presented. The ability to produce somatic embryos offers us the opportunity to use embryo-tissue for the production of embryogenic callus.