Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid     

Protoplast isolation, culture, and fusion protocols for kenaf (Hibiscus cannabinus)

Protoplast isolation and culture protocols were developed for ten cultivars of Hibiscus cannabinus L. (kenaf). Leaves from seedling lines maintained in vitro were used as donor tissues. Optimal cell wall digestions were achieved with a combination of cellulysin (1.0%) and macerase (0.5%). Average yields ranged from 0.9×10⁵ to 5.9×10⁶ protoplasts g fw^-1 leaf tissue with viability estimates ranging from 53% to 87%. This protocol was ineffective for leaf tissue taken from plants grown in vivo. Protoplasts harvested from plantlets maintained in vitro produced rapidly growing calluses when plated in semi-solid medium after an initial culture in liquid medium. First cell divisions were observed within four to six days after initial culture in medium containing plant growth regulators 2,4-dichlorophenoxyacetic acid (1.4 M) and kinetin (13.8 M). An electrofusion protocol which did not significantly reduce protoplast viabilities was developed for kenaf protoplasts. The maximum fusion frequency (4.6%) was obtained with an electrofusion voltage of 2.0 kV cm^-1.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - FDA fluorescein diacetate - MS Murashige and Skoog - NAA 1-naphthaleneacetic acid - PGRs plant growth regulators - SCL seed clonal line     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and callogenesis in tissue culture of floral organs of the genus <Emphasis Type="Italic">Iris</Emphasis> (Iridaceae)

We tested the differentiation and morphogenetic capacity of floral organs of Iris ensata, I. setosa, and I. sanguinea cultured in vitro. Organogenesis through direct formation of shoots from explants, callogenesis, and floral organogenesis were demonstrated in I. ensata callus culture in vitro. These processes depended on the plant species and on the content of phytohormones in the medium. Adventitious shoots proved to develop on the basal part of the perianth tube and on the apical part of the ovary, while roots were not formed. Direct organogenesis was induced by the following phytohormones: -naphthylacetic acid and 6-benzylaminopurine for I. ensata and 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine for I. setosa and I. sanguinea; while callogenesis was induced by 2,4-dichlorophenoxyacetic acid. The obtained data indicate that development of adventitious structures from iris floral organs requires the presence of 6-benzylaminopurine in the growth medium.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 2, 2005, pp. 174–179.Original Russian Text Copyright © 2005 by Boltenkov, Zarembo.     

Somatic embryogenesis and plant regeneration from isolated protoplasts of Lavatera thuringiaca

Embryogenic cell suspensions of Lavatera thuringiaca L. were established from leaf petiole and shoot regeneration was achieved when cells were plated on medium without growth regulators. We tested three methods for protoplast culture, isolated from a one-year old embryogenic cell suspension, to determine the best conditions for L. thuringiaca protoplast culture and shoot regeneration. The highest protoplast plating efficiency was obtained with the agaroseembedded method, reaching 30%, while the nursing culture method gave 5% when the protoplasts were plated over Whatman paper No. 2. However, the same nursing culture failed to produce protoplast-derived microcalluses when the protoplasts were plated on a nitrocellulose filter. The liquid thin layer method gave the lowest plating efficiency with only 0.5%. Shoot regeneration from protoplast-derived microcalluses was achieved in two steps; first, globular embryo development was favored in medium low in auxin (2,4-d and BA at 0.01 and 0.05 mg 1^-1, respectively), second, the globular embryos further differentiate into shoots in medium without growth regulators or in medium containing GA₃ (0.5 to 1.0 mg 1^-1).Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - IBA indole-3-butyric acid     

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume<Emphasis Type="Italic"> Astragalus melilotoides</Emphasis> Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74±0.6×10⁵/g FW) and viability (87.07±2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3–7 days following culture initiation. The highest division frequency (9.86±0.68%) and plating efficiency (1.68±0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l -naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3±4.1%) and organogenesis (21.6±0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.Abbreviations BA: 6-Benzylaminopurine - CH: Casein hydrolysate - CPW: Cell protoplast wash - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FDA: Fluorescein diacetate - IBA: Indole-3-butyric acid - KIN: Kinetin - MES: 2-(N-morpholino) Ethanesulphonic acid - NAA: -Naphthaleneacetic acidCommunicated by A. Altman     

Involvement of ethylene on growth and plant regeneration in callus cultures of Heliconia psittacorum L.f.

The level of ethylene accumulated in morphogenic callus cultures of Heliconia psittacorum L.f. was only one quarter that of non-morphogenic cultures. The rate of ethylene production in the morphogenic callus cultures during early stages of differentiation of protocorm-like bodies leading to plantlet regeneration was 10-fold higher than that during callus proliferation. In cultures sealed with gastight serum caps, fresh weight gain was reduced 2-to 3-fold compared to those that were closed with Kaputs. Treatment with 1-aminocyclopropane-1-carboxylic acid ( 100 M) caused complete inhibition of plant regeneration from the morphogenic callus on subsequent culture under inductive conditions. Silver nitrate and aminoethoxyvinylglycine also reduced plant regeneration. These results indicate that while high levels of ethylene were inhibitory, a low level of endogenous ethylene production may be necessary during the plant regeneration phase in callus cultures of Heliconia.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - AC activated charcoal - ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BM basal medium - CH casein hydrolysate - DM development medium - MM maintenance medium - PLB protocorm-like body     

Induction of microspore embryogenesis in Camellia japonica cv. Elegans

Three methods of microspore culture were tested for the induction of microspore embryogenesis in Camellia japonica L. cv. Elegans. Culture was performed on 17 different media consisting of Murashige and Skoog (MS) and N6 basal media with different combinations of carbon, growth regulators, serine and glutamine. Microspore suspensions plated over solid MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 0.5 M kinetin, with sucrose (MS6) or glucose (MS9) were seen as the best culture conditions for induction of embryogenesis. The development of microspore derived proembryos was obtained in MS medium supplemented with 2.2 M N⁶-benzyladenine (MS10) and reached the highest level when the microspores were cultured in MS6 inducing medium. The development of microspore-derived embryos ceased at the maturation stage.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid     

Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets

Protoplasts were isolated from field and in vitro-grown leaves, cotyledons and cell suspension cultures (of ovule callus origin) of the scion apple cultivars Starkrimson, Rainier, Qiujin and Liaofu. Fast-growing calluses were obtained from leaf, cotyledon and cell suspension derived protoplasts of the four genotypes. The best proliferation responses were obtained from cell suspension protoplasts. For all genotypes tested, nodular calluses were obtained from protoplasts that had originally been cultured on K8P medium, but only those of cultivar Starkrimson underwent organogenesis. In this cultivar shoot buds were produced on callus derived from both cotyledon and cell suspension protoplasts and complete plants. This is the first example of whole plant regeneration from protoplasts isolated from an undifferentiated tissue in apple.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA 3-indole acetic acid - IBA 3-indole butyric acid - LH lactalbumin hydrolysate - MS Murashige & Skoog (1962) - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - VC L(+) ascorbic acid     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid     

High frequency somatic embryogenesis in Coffea canephora

Leaf explants of Coffea canephora (P. ex Fr.) produced a friable yellow callus when they were cultured on a conditioning basal medium with 2.2 M 2,4-D, 2.4 M IBA and 9.8 M 2iP for 4 weeks then on an induction basal medium with 4.4 M 2,4-D and 17.8 M BA for 10 weeks. This calus could be maintained by means of regular subcultures or it could give rise to somatic embryos depending on the culture medium. Cytological studies documented somatic embryogenesis and embryo development.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - 2iP 2-isopentenyladenine - MS Murashige & Skoog medium - NAA -naphthaleneacetic acid - NPR nucleoplasmic ratio - PGR plant growth regulator     

Cell differentiation in protoplast cultures from embryogenic callus ofAbies alba L.

Protoplasts were isolated from embryogenic callus ofAbies alba L. which originated from immature seeds. The protoplasts were immobilized in alginate beads in order to follow the development of single protoplasts.Surrounding culture medium was modified from Kao and Michayluk (1975). After cell wall regeneration subsequent cell divisions lead to the formation of colonies showing an early differentiation of small meristematic cells and large vacuolated suspensor-type cells.Abbreviations 6-BA N⁶-benzyladenine - IAA 3-indoleacetic acid (potassium salt) - KM Kao and Michayluk (1975) - MES 2-(N-morpholino)ethanesulfonic acid - NAA 1-naphthalene acetic acid (sodium salt) - PVP Polyvinylpyrrolidone - SH Schenk and Hildebrandt (1972) - Tween 80 Polyoxyethylene- sorbitan - monooleate - WPM (woody plant medium), Lloyd and McCown (1981)     

Somatic embryogenesis and callus production from cotyledon explants of Eastern black walnut

Starting at 8 weeks and continuing until 23 weeks (nut drop) after anthesis,1 m² explants from cotyledons of immature seeds were extracted from Juglans nigra fruits. Explants were placed on Woody Plant Medium with 1 g l^-1 casein hydrolysate and 30 g l^-1 sucrose. The explants remained in light for 4 weeks on primary media containing a 3×3 factorial of 0.05, 0.5, or 5.0 M thidiazuron (TDZ) and 0.1, 1.0, or 10.0 M 2,4-d. Explants were transferred to a secondary medium containing no plant growth regulators and incubated in darkness for 11 weeks. The greatest number of somatic embryos was produced 8, 10, and 12 weeks after anthesis from explants on media with 0.5 or 5.0 M TDZ and 0.1 or 1.0 M 2,4-d. Explants produced the greatest callus volume and dry weight 10, 12, and 14 weeks after anthesis. Throughout the study, callus generally increased with increasing concentrations of both TDZ and 2,4-d.Abbreviations BA 6-benzyladenine - captan 3a,4,7,7a-tetrahydro-2-[(trichloromethyl)thio]-1H-isoindole-1,3(2H)-dione - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - Physan n-alkyl- dimethyl-benzyl ammonium chlorides and n-alkyl-dimethyl-ethylbenzyl ammonium chlorides - TDZ-thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

Development and germination of Abies alba somatic embryos

Mature zygotic embryos of Abies alba mull were placed on a modified MCM medium (basal medium-BM) with 2.2 M benzyladenine and 2.3 M kinetin to induce embryogenic suspensor masses (ESM). These ESM proliferated on induction medium supplemented with 0.2 M 2,4-dichlorophenoxyacetic acid. From 61 ESM lines induced, 36 are still in culture after 2 years, of which 18 show embryogenic potential indicated by spontaneous formation of globular somatic embryos on the proliferation medium supplemented with 500–1000 mg l^-1 casein hydrolysate and 500 mg l^-1 l-glutamine. ESMs from cell line 2/56 were conditioned 1 week on BM with 58 mM sucrose and 10 g l^-1 activated charcoal for maturation of somatic embryos. Maturation was achieved on BM containing 20 M (±)cis-trans-abscisic acid in combination with 111 mM maltose. Organic nitrogen supplements improved the proliferation rate of cell line 2/56 as well as the maturation and vitality of the somatic embryos. Partial drying was necessary for subsequent root development. Plantlets with a root, primary needles and a terminal bud developed on BM when a combination of 30 mM sucrose and 50 mM maltose was provided as carbon source.Abbreviations BM basal medium - BA benzyladenine - ESM embryogenic suspensor mass - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - l-gln l-glutamine - ABA (±) cis-trans-abscisic acid     

Plant regeneration via somatic embryogenesis in ginger

Embryogenic callus cultures of ginger were induced from young leaf segments taken from in vitro shoot cultures. Among the four auxins tested in Murashige & Skoog medium, dicamba at 2.7 M was most effective in inducing and maintaining embryogenic cultures. Efficient plant regeneration was achieved when embryogenic cultures were transferred to Murashige & Skoog medium containing 8.9 M benzyladenine. Histological studies revealed various stages of somatic embryogenesis characteristic of the monocot system. The in vitro-raised plants have been established in soil.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

In vitro plantlet regeneration of Ocotea catharinensis,an endangered Brazilian hardwood forest tree

A successful system of somatic embryogenesis is described for the forest tree Ocotea catharinensis Mez., which used mature zygotic embryo explants cultured on a modified Murashige and Skoog (MS) medium with activated charcoal, at 25°C in the dark. A medium composed of MS supplemented with 2% (w/v) sucrose, 0.3% (w/v) activated charcoal (AC), 362 M 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.8% (w/v) Technical Agar Grade III was used for multiplication of embryogenic cultures. Development up to the globular-stage was achieved using Lloyd and McCown woody plant medium (WPM) with 2.0% sucrose, 0.3% AC, 181 M 2,4-d and 0.8% Technical Agar Grade III. Significant effects of media pH on differentiation of early pro-embryogenic Ocotea cell aggregates were found. Low pH of media (ca. 3–4) appeared to prevent differentiation of proembryogenic cell aggregates whereas higher pH levels (ca. 5–5.5) favoured the formation of globular structures. Once globular structures formed, they developed further to form cotyledonary somatic embryos, under the same set of culture conditions. Successful conversion of these somatic embryos to plantlets was achieved after culture on a medium composed of 1/2-strength WPM (minerals only) with 2% sucrose, 0.3% AC, 0.8% Technical Agar Grade III and 90.5 M 2,4-d, followed by transfer to a medium composed of 1/2-strength WPM (minerals only) with 2% sucrose, 0.8% Technical Agar Grade III and 0.905 M 2,4-d and 1.4 M gibberellic acid, in a 16-h photoperiod regime.     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Plant regeneration from mesocotyl callus of Hordeum vulgare L.

Callus tissue was induced on barley mesocotyl explants of germinated seven-day-old seedlings on MS medium supplemented with 2,4-D or 2,4,5-T in high concentrations. Two morphologically different tissue cultures were maintained in vitro for a long time: a callus tissue without organogenesis and a culture with high rhizogenic capacity. Shoots and plantlets were generated when the auxin-media induced callus was transferred to medium supplemented with 3 M TIBA. In 62% of cultures, during the first five subcultures, four to twentyeight plants per single mesocotyl were obtained. Some cultures produced shoots even in the 9th subculture, being in culture for nearly 14 months. The largest number of plants obtained per one mesocotyl was forty. Plantlets rooted well on MS with 5.7 M IAA and survived transplantation to soil in high percentages.Abbreviations IAA indole-3yl-acetic acid - BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog (1962) medium