Changes in isoperoxidases during shoot formation in tobacco callus

Shoot formation in tobacco (Nicotiana tabacum L.) callus is accompanied by an increase in peroxidase activity which takes a form similar to a sigmoid curve. The "stationary" phase coincides with the period of organ formation. Characteristic changes in isoperoxidase pattern are found in the shoot-forming part of the callus. These changes are different from those in the nonshoot-forming part or in gibberellin-treated tissue, which does not form shoots.     

In vitro plant regeneration of Mammillaria elongata normal and cristate forms

In the normal form of Mammillaria elongata, shoots were regenerated in vitro, through callus, from tubercle explants excised from the upper part of the branch and cultured on Murashige and Skoog medium (MS) with 1.07 μM α-napthaleneacetic acid (NAA) and 22.20 μM 6-benzylaminopurine (BA). A high percentage of tubercles explants of the M. elongata cristate form, excised from the tip of the branch and cultured on MS with 0.54 μM NAA and 0.44 μM BA or 1.07 μM NAA, responded by initially forming an inflated cristate shoot, which gave cristate and normal shoots, without callus intervention, when transferred on basal MS. Callus formed on cristate tubercles explants gave both cristate and normal shoots when transferred onto basal MS. Normal and cristate shoots were rooted in vitro on MS with 9.84 μM or 0.98 μM indole-3-butyric acid, respectively, and established ex vitro. In both normal and cristate form, the differential response appeared to be associated with the site of the explant excision. The formation of shoots was influenced by the season of culture; i.e., explants excised in October had a higher shoot formation rate than those excised February. This revised version was published online in June 2006 with corrections to the Cover Date.     

Somatic embryogenesis in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) flower stem cultures

In the present study, the procedures for induction of somatic embryogenesis (SE) in an in vitro culture of the tulip have been developed. SE was initiated on flower stem explants isolated from “Apeldoorn” bulbs during their low-temperature treatment. Bulbs had not been chilled or had been chilled for 12 or 24 weeks at 5°C. The explants were cultured with exogenous auxins 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3,5,6-trichloropicolinic acid (Picloram), α-naphthaleneacetic acid (NAA) at 1–100 μM and cytokinins: benzyladenine (BA) and zeatin (ZEA) at 0.5–50 μM. Increase in auxin concentrations caused an intensive enlargement of the explant parenchyma, which changed into homogenous colorless callus. On the same media, vein bundles developed into yellowish, nodular callus. Picloram was more efficient in inducing the formation of embryogenic nodular callus than 2,4-D, whereas the latter stimulated formation of colorless callus. The base of the lower part of the flower stem isolated from bulbs chilled for 12 weeks proved to be the best explant for callus formation. The highest number of somatic embryos was produced on medium with 25 μM Picloram and 0.5 μM BA. Development of adventitious roots was noticed in the presence of 2,4-D. Globular embryos developed into torpedo stage embryos under the influence of BA (5 μM) and NAA (0.5 μM). Morphological and anatomical data describing development of callus and somatic embryos are presented.     

Effects of cytokinin on adventitious root formation in callus cultures ofVigna unguiculata (L.) walp

Summary Yellowish compact callus, induced from cowpea hypocotyls on Murashige and Skoog(MS) medium (1962) containing 0.2 mg/l(0.93 μM) kinetin and 0.4 mg/l (1.81 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), was subcultured on MS medium containing cytokinin alone, auxin alone, or auxins plus cytokinins in order to determine the effect of cytokinins on root organogenesis in callus cultures. The callus actively proliferated on the same medium but did not show any organogenic activity macroscopically as well as microscopically. On medium with N⁶-benzyladenine (BA) and 1-naphthaleneacetic acid (NAA), the yellowish compact callus first changed to pale green compact callus and then many green spots appeared on its surface under light culture. But the yellowsih compact callus remained yellowish and white spots appeared on its surface in dark culture. These spots gradually became white nodular structures. Adventitious root formation from the nodular structures occurred not only on the same medium, but also on medium with either auxin or cytokinin but not both. Yellowish compact callus on medium with auxin alone was transformed to yellowish friable callus, which did not develop adventitious roots. The yellowish friable callus could gain rhizogenic activity only after morphological modification to pale green compact callus on medium with auxin plus cytokinin. The modified callus did not form adventitious roots on medium with auxins but only with cytokinins. Therefore, it is suggested that cytokinins have stimulating effects on root formation from callus that previously did not show rhizogenic activity on medium with auxins alone. In addition, the rhizogenic potential of cowpea callus was discriminated from that of leaf explants, which formed adventitious roots directly on medium with auxin alone.     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.     

Histological observations of morphogenesis in petiole derived callus of Amorphophallus rivieri Durieu in vitro

The initiation and development of somatic embryos and organogenic shoots and corm-like structures (CLSs) from petiole-derived calli of Amorphophallus rivieri Durieu were observed histologically. The petioles were cultured on Murashige and Skoog (MS) medium supplemented with 5.37 μM α-naphthaleneacetic acid (NAA) and 4.44 μM N⁶-benzylaminopurine (6-BA) for callus induction. The shoot and corm organogenesis occurred from the compact calli when they were transferred to a medium containing 0.54 μM NAA and 4.44 μM 6-BA. A combination of 13.57 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88 μM 6-BA or 24.18 μM NAA and 6.66 μM 6-BA was optimum for induction of somatic embryos, which failed to produce plantlets because of their structural abnormalities. Shoot regeneration predominantly happened through organogenesis although somatic embryogenesis infrequently occurred. The subepidermal cells of the compact callus converted to competent cells and started divisions, which resulted in formation of the meristemoids. The meristemoid cells continued division to develop into bud primordia. Subepidermal cells could also form the globular structures. Subsequently, these globoids developed into CLSs from which plantlets regenerated during subculture. Meanwhile, the CLSs were capable to form cormels, which could be a promising way for the propagation of A. rivieri.     

Low activity of amine-oxidases and accumulation of conjugated polyamines in disfavour of organogenic programs in Chrysanthemum leaf disc explants

Foliar discs (8 mm diameter) from expanding leaves of the middle part of vegetative shoots of Chrysanthemum morifolium Ramat raised in vitro were induced to form directly on specific media in vitro either roots or vegetative buds, or callus. The budding programme, on its specific medium, was deviated to callus formation by the addition of 2 mM β-OH-E (β-OH-ethyldrazine, an inhibitor of diamine oxidase). Conversely vegetative buds instead of callus were formed on the callus medium in the presence of 2 mM DFMO (difluoromethylornithine, an inhibitor of ornithine decarboxylase). Callus formation was characterized by high accumulation of free and particularly conjugated polyamines (PA), very low or undetectable activities of diamine- and polyamine oxidases, and transglutaminase. DFMO-deviation of callus initiation in favour of bud formation lowered the accumulation of PA and increased the activity of amine-oxidases. The high catabolism of PA in the organogenic (rooting, budding) programs was questioned as to its role in developmental processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Callus formation and plant regeneration in various <Emphasis Type="Italic">Lilium</Emphasis> species and cultivars

Summary 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.     

The effects of low levels of chloramphenicol and methotrexate on somatic embryogenesis inCitrus

Summary The effects of the antibiotics methotrexate and chloramphenicol on somatic embryogenesis inCitrus were evaluated. Relatively low levels (0.1 to 1.0 μg/ml) of these antibiotics did not inhibit embryo production from undeveloped ovules of ‘Key’ lime [C. aurantifolia) (Christm.) Swing.]. Surprisingly, both antibiotics induced the formation of embryogenic callus in these cultures. This is usually a rare event in cultures of undevelopedCitrus ovules, and ‘Key’ lime is especially recalcitrant. The effects of these antibiotics on embryogenic callus appeared to be limited to the induction stage, because there was no consistent effect, either stimulatory or inhibitory, on established, lines of embryogenic callus. Florida Agricultural Experiment Station Journal Series No. 8958. This research was supported in part by a grant to Moore and Cline from the Competitive Grants Office of the SEA, USDA (85-CRCR-1-1623).     

Highly-efficient somatic embryogenesis from cell suspension cultures of phalaenopsis orchids by adjusting carbohydrate sources

Summary The influences of various carbohydrate sources, dried yeast (DY), and 6-benzylaminopurine (BA) were estimated on growth and development of shoot tip-derived suspension cells of phalaenopsis orchid. Among the carbohydrates tested on Doriataenopsis cultured on gelled medium, glucose at 58.4 mM gave the highest efficiency of protocorm-like body (PLB) formation. Maltose and sorbitol only induced PLB formation without callus proliferation. Sucrose induced comparable callus proliferation to glucose but without PLB formation. In contrast, fructose resulted in half the amount of callus proliferation as occurred with glucose. Lactose was an inadequate carbon source as neither PLB formation nor callus proliferation occurred. DY enhanced cell proliferation at 0.1–1gl⁻¹ but inhibited both cell proliferation and PLB formation at 10gl⁻¹. Low BA (0.4 μM) slightly increased callus proliferation but inhibited PLB formation. Only one treatment, sucrose and 1 gl⁻¹ DY, yielded a small number of plants. For suspension cultures of Phalaenopsis Snow Parade and P. Wedding Promenade, PLB formation was most efficiently induced by sucrose at 29.2 mM for P. Snow Parade and 14.6 mM glucose for P. Wedding Promenade. Histological observation revealed that cells in suspension culture developed into plants through the same developmental proess as germinating seeds.     

Morphogenesis and regeneration from stomatal guard cell complexes of cotton (Gossypium hirsutum L.)

 The development of a regeneration system from cotton stomatal guard cells directly on epidermal strips is described. The most important factors affecting embryogenic callus initiation in both of the varieties tested (Coker 312 and 315) were the source of the epidermal tissue, including plant age (4–5 months old), the developmental stage of the flower (opening flower stage) from which bracts were obtained, the composition of the culture medium and light irradiance. The flower developmental stage was critical for callus formation, which was observed only from bracts obtained from opening flowers. In addition, epidermal strips excised from the bract basal region were more responsive in culture than those obtained from the top region. Improved callus initiation was obtained on epidermal strips which had their cuticle in contact with the culture medium. Light irradiance was a limiting factor for embryogenic callus formation, which was observed only in calluses cultured under the lower light irradiance (15.8 μmol m^–2 s^–1). Somatic embryogenesis was observed on callus cultures subcultured consecutively to a culture medium containing naphthalene acetic acid (10.7 μM) and isopentenyladenine (4.9 μM). Histodifferentiation of somatic embryos was improved on a medium containing naphthaleneacetic acid (8.1 μM)+isopentenyladenine (2.5 μM) and abscisic acid (0.19–0.38 μM). Somatic embryo germination and plantlet development were obtained using established protocols with few modifications. On average, one fully developed plant was obtained from the culture of circa 100 epidermal strips in both cultivars. Received: 19 May 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Induction of adventitious buds in cultured petal explants ofChelidonium majus L.

Petal explants ofChelidonium majus L. (Papaveraceae) formed noteworthy adventitious buds without any intermediate callus when cultured under appropriate conditions. Bud formation was favored by combinations of 1–2 mg/l indoleacetic acid (IAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–0.5 mg/l kinetin (K). In the present study, neither bud formation nor callus formation occurred in cultures of excised leaves. A histological study revealed that adventitious bud formation occurred only in single epidermal layers of petals, while several subepidermal parenchyma layers did not join in its formation. Activation zones arising from the epidermis underwent intense cell divisions to initiate buds on the epidermal surface. These buds later turned green in color, developing into shoots which eventually grew into plantlets after root formation.     

Determination in plant cells

The possibility that some of the variation in callus cultures involves epigenetic changes is examined in cultures established from the hypoootyls and roots ofEuphorbia heterophylla. It is shown that the responses of the cultures are affected by the light regimes under which they are grown and that in the dark and under short photoperiods, there are differences between the two types of culture with respect to pigmentation, auxin requirement, capacity to regenerate buds and roots and in certain isozyme patterns, whereas the two cultures are similar from the first passage under continuous light. However, these differences are only maintained for 2–3 passages, after which the root callus becomes similar to the hypoc otyl callus. Evidence is presented that these differences between cultures are epigenetic. Callus cultures established from the apical meristems of shoots and roots ofE. hetero phylla show similar differences to those observed between hypocotyl and root cultures and these differences are also lost after 3 passages. These results indicate that the cells of apical meristems are not totally uncommitted, but are determined as ‘shoot’ and ‘root’ meristem colls, respectively. The practical importance of a better understanding of epigenetic effects in plant cells is strassed.     

Direct and callus-mediated protocorm-like body induction from shoot-tips of <Emphasis Type="Italic">Dendrobium chrysotoxum</Emphasis> Lindl. (Orchidaceae)

An efficient method of mass propagation of Dendrobium chrysotoxum Lindl. was developed using a shoot-tip culture system. Both direct and callus-mediated formation of protocorm-like bodies (PLBs) occurred from the basal cut surface of explants. Frequency of callusing was best in the presence of 2 μM thidiazuron (TDZ) or N⁶-benzylaminopurine (BAP). The callus exhibited complete hormone autonomy for growth and differentiation of PLBs and was maintained for 18 months without any exogenous growth regulators, an aspect important for minimising somaclonal variation. However, the rate of callus growth and PLB formation varied with application of cytokinin and auxin. In addition, the callus exhibited a differential sensitivity to the exogenous cytokinins. While BAP promoted callus growth and PLB differentiation, TDZ was inhibitory to callus mediated PLB formation and caused extensive necrosis of callus. Although α-naphthaleneacetic acid (NAA) had no significant effect on the induction of callus, subsequent growth was best in its presence. Using a 3-month subculture period, a 69-fold increase in callus weight was achieved with 0.5 μM NAA, while as many as 133 PLBs could be obtained per 100 mg callus in the presence of 1 μM NAA. For direct PLB formation, the optimum cytokinin dosage was dependent upon the type of cytokinin used. While TDZ was most effective at a concentration of 1 μM (15 PLBs per explant), for similar PLB yield the application of 8 μM BAP was essential.     

Thidiazuron-induced high-frequency shoot organogenesis from leaf-derived callus of ia medicinal climber, <Emphasis Type="Italic">Tylophora Indica</Emphasis> (Burm. F.) merrill

Summary Tylophora indica (Burm. f.) Merrill is a threatened medicinal climber distributed in the forests of northern and peninsular India. An efficient and reproducible protocol for high-frequency callus regeneration from immature leaf explants of T. indica was developed. Organogenic callus formation from immature leaf pieces was obtained by using Murashige and Skoog (MS) medium supplemented with 7 μM 2,4-dichlorophenoxyacetic acid and 1.5 μM 6-benzyladenine. On this medium 92% explants produced callus. The optimal hormone combination for plantlet regeneration was 8 μM thidiazuron, at which shoot regeneration was obtained from 100% of the cultures, with an average of 66.7 shoots per culture. Histological studies of the regenerative callus revealed that shoot buds were originated from the outermost regions. For root formation, half-strength MS medium supplemented with 3 μM indole-3-butyric acid was used. Plants were transferred to soil, where 92% survived after 3 mo. of acclimatization.     

Effects of genotype, explant source, and plant growth regulators on indirect shoot organogenesis in Dieffenbachia cultivars

The capacity for indirect shoot organogenesis of leaf and root explants of four Dieffenbachia cultivars were examined on a modified Murashige and Skoog (MS; Physiol Plant 15:473–495, 1962) medium supplemented with different plant growth regulators in 112 combinations. Callus formation was only observed from leaf explants on MS supplemented with 1–10 μM thidiazuron (TDZ) and 0.5–1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) regardless of cultivars. The combination of 5 μM TDZ and 1 μM 2,4-D resulted in the greatest callus formation frequency among the four cultivars tested. Significant differences in callus and shoot formation from leaf explants were also observed among cultivars. Cultivars Camouflage, Camille, Octopus, and Star Bright produced green nodular, brown nodular, yellow friable, and green compact calli with corresponding maximum callus formation frequencies of 96%, 62%, 54%, and 52%, respectively. A maximum of 6.7 shoots/callus was observed in cv. Camouflage, followed by cvs. Camille and Star Bright at 3.7 and 3.5, respectively. Calli of cv. Octopus displayed no capacity for shoot organogenesis. Regardless of cultivar, callus formation was not observed on root explants. Regenerated shoots were successfully acclimatized in a shaded greenhouse condition with 100% survival.     

Predicting the external formation of a bone fracture callus: an optimisation approach

The formation of a fracture callus in vivo tends to form in a structurally efficient manner distributing tissues where mechanical stimulus persists. Therefore, it is proposed that the formation of a fracture callus can be modelled in silico by way of an optimisation algorithm. This was tested by generating a finite element model of a transversal bone fracture embedded in a large tissue domain which was subjected to axial, bending and torsional loads. It was found that the relative fragment motion induced a compressive strain field in the early callus tissue which could be utilised to simulate the formation of external callus structures through an iterative optimisation process of tissue maintenance and removal. The phenomenological results showed a high level of congruence with in vivo healing patterns found in the literature. Consequently, the proposed strategy shows potential as a means of predicting spatial bone healing phenomena for pre-clinical testing.     

Developmental steps in acquiring competence for shoot development in <Emphasis Type="Italic">Arabidopsis</Emphasis> tissue culture

Arabidopsis shoots regenerate from root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium (CIM) followed by incubation on a cytokinin-rich shoot induction medium (SIM). During CIM preincubation, root explants acquire competence to respond to shoot induction signals. During CIM preincubation, pericycle cells in root explants undergo cell divisions and dedifferentiate, losing the expression of a pericycle cell-specific marker. These cells acquire competence to form green callus only after one day CIM preincubation and to form shoots after 2–3 days CIM preincubation. Reversible DNA synthesis inhibitors interfered with the acquisition of competence to form shoots. Genes requiring CIM preincubation for upregulation on SIM were identified by microarray analysis and included RESPONSE REGULATOR 15 (ARR15), POLYGALACTURONASE INHIBITING PROTEIN 2 (PGIP2) and WUSCHEL (WUS). These genes served as developmental markers for the acquisition of competence because the CIM preincubation requirements for ARR15 and PGIP2 upregulation correlated well with the acquisition of competence to form green callus, and the CIM preincubation requirements for WUS upregulation matched those for shoot formation. Unlike ARR15, another cytokinin inducible, A-type ARR gene, ARR5, was upregulated on SIM, but the induction did not require CIM preincubation. These findings indicate that competencies for various events associated with shoot regeneration are acquired progressively during CIM preincubation, and that a set of genes, normally upregulated on SIM, are repressed by a process that can be relieved by CIM preincubation.