Plant regeneration from callus cultures of Durum and emmer wheat

Callus cultures were initiated from isolated mature embryos of Triticum turgidum L. Thell ssps durum and dicoccum on a basal medium supplemented with 2,4-D, 2,4,5-Cl₃POP or 2,4-D+CM. Shoot bud regeneration was observed on 2,4,5-Cl₃POP medium. In both the cultivars of durum, further development of shoot buds occurred on transfer of tissues to basal medium whereas in dicoccum basal medium supplemented with coconut milk or coconut milk with NAA (0.2 mg/l) was necessary. The regenerated shoot buds were induced to root on basal medium supplemented with NAA. The in vitro obtained plants were transferred to soil and successfully grown to maturity. Chlorophyll variants were observed among the regenerated plants of dicoccum.Abbreviations BA benzyladenine - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,iP 6---dimethylallylamine purine - IAA indoleacetic acid - NAA -naphthalene acetic acid - Kn kinetin - 2,4,5-Cl₃POP 2,4,5-trichlorophenoxypropionic acid - MS modified Murashige and Skoog's medium - RH relative humidity - Z zeatin     

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata Crandon, NC-5339 and P. nigra Betulifolia × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula Erecta) and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.Abbreviations BA benzyladenine - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - NC-XXXX North Central Forest Experiment Station accession number assigned to hybrid poplar clones.     

Growth and differentiation of callus cultures of Pinus

Segments of hypocotyl and cotyledons of aseptically-grown seedlings of Pinus strobus L. (white pine) and P. echinata Mill (shortleaf pine) were used as explants for establishing tissue cultures. Growth and differentiation of callus were studied on a modified Murashige and Skoog's medium containing nutrients and plant growth regulators. Meristems below the surface of callus tissue of P. strobus could be induced on media supplemented with -naphthaleneacetic acid alone or in combination with certain other plant growth regulators. Occasionally, differentiation of shoot buds also occurred on callus cultures. These shoot buds could be grown in vitro but roots did not develop.Abbreviations ABA abscisic acid - BA 6-Benzyl-aminopurine - 2-ip N⁶-(²-isopentanyl)-adenine - GD Gresshoff and Doy's medium - GE Gamborg and Eveleigh's medium - MS Modified Murashige and Skoog's medium - NAA -naphthaleneacetic acid - SC Sommer and Caldas' medium - TIBA 2,3,5-Triiodobenzoic acid     

Release and crystallization of berberine in the liquid medium of Thalictrum minus cell suspension cultures

Cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. were found to produce a large amount of berberine (400–800 mg/l) when 5–10 M 6-benzyladenine was added to Linsmaier and Skoog's medium containing 100 M 1-naphthaleneacetic acid. Most of the berberine produced was released continuously from the cells into the liquid medium, and an excess of berberine crystallized as its nitrate in the medium. When the cells were cultured in a modified LS medium containing 20 mM KNO₃ and 40 mM NH₄Cl in place of 20.6 mM NH₄NO₃ as nitrogen source, most of the alkaloid crystallized to form berberine chloride instead of nitrate. Minor alkaloids, thalifendine and magnoflorine, were also isolated from the medium and identified.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzyladenine     

Growth rate and methane affinity of a turbidostatic and oxystatic continuous culture ofMethylococcus capsulatus (Bath)

Summary A turbidostatic and oxystatic fermentation system was used to study the growth kinetic ofMethylococcus capsulatus (Bath). Dissolved oxygen and methane concentrations were measured continuously with membrane inlet mass spectrometry. The specific growth rate was found to increase from 0.25 h^–1 to 0.37 h^–1 and the saturation constant for methane was found to decrease from 71 M to 1.3 M as the copper content of the medium was varied from a very low to a high value.     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Improved efficiency of plant regeneration from protoplasts of eggplant Solanum melongena L.

Eggplant (Solanum melongena L.) mesophyll protoplasts were obtained from in vitro growing plants of line 410 and cv. Classic. Relatively high (15%) plating efficiency was achieved using petri dishes with alternate quadrants containing reservoir medium (R medium + 1% activated charcoal) and culture medium. Shoot regeneration occurred within 6 weeks following initiation of protoplast culture.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan Israel, No. 1164-E, 1984 Series.     

Rapid micropropagation for Mucuna pruriens f. pruriens L.

A rapid micropropagation system was developed for Mucuna pruriens f. pruriens using explants from 1-week-old aseptically grown seedlings. Multiple shoot regeneration occurred following an initial callus growth on Revised Tobacco (RT) medium supplemented with 2.7 M NAA and 9.8 M 2iP. Maximum number of shoot regeneration was achieved only from seedling explant 6 to 7 days old. More than 90% of the regenerated shoots could be rooted on half-strength liquid RT medium supplemented with 2.7 M NAA. Plantlets readily adopted to greenhouse conditions. This system provides a new tool for micropropagation of Mucuna pruriens f. pruriens, an important medicinal plant.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - 2iP 2-isopentenyladenine - AdS adenine sulphate - NAA alpha-naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - RT Revised Tobacco medium (Kaul and Staba, 1968)     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Micropropagation of Uniola paniculata L.

Uniola paniculata L. is a major sand-dune stabilizing grass which is being utilized to prevent shoreline erosion. In vitro cultured caryopses of U. paniculata produced callus on MS medium supplemented with 22.5 M 2,4-D, 4.4 M BA and 87.6 mM sucrose. Shoot induction occurred after these calli were inoculated onto the same medium without 2,4-D. Rooting of in vitro-derived shoots occurred when transferred to a one-half strength MS medium containing 43.8 mM sucrose and 14.7 M IBA. Plantlets were planted after the roots reached a length greater than 20 mm.Abbreviations BA N-(phenyl-methyl)-lH-purine-6-amine - IBA lH-indole-3-butanoic acid - MS Murashige and Skoog (1962) - 2,4-D (2,4-dichlorophenoxy) acetic acid - TC agar tissue culture agar, (K.C. Biologicals, Lenexa, KS) - Subdue methaxyl:N-(2,6-dimethylphenyl)-N-(methyoxyacetyl) alanine methyl ester (CIBA- GEIGY)     

Anther culture of some perennial triticeae

Three field grown Agropyron spp. (crested wheatgrasses) and two Thinopyrum spp. (intermediate and tall wheatgrasses) were evaluated for anther culture response. Hormonally modified potato extract and 85D12 media induced pollen embryogenesis. Modified Murashige and Skoog media were tested for their effects on callus proliferation and plantlet regeneration. Callus induction frequency and plantlet production were highest (25.0% and 45.8%, respectively) for Thinopyrum ponticum (2N=70) (tall wheatgrass). One-hundred and nine albino plantlets were produced from T. ponticum Jose both by direct regeneration on 85D12 medium and through a callus phase from potato extract media. This is the first report of plantlet production from anther culture of a Triticeae perennial forage grass. Further experimentation with environmental and cultural conditions may result in the production of green plantlets.Abbreviations MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-ip 2-isopentenyladenosine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid Cooperative investigations of the USDA-Agricultural Experiment Station and the Utah Agricultural Experiment Station, Logan, UT 84322. Approved as Journal Paper No. 3596     

Plant regeneration of Oncidium varicosum (Orchidaceae) by means of root tip culture

The effects of an auxin (NAA) and a cyto kinin (BA), as well as those of peptone, thiamin and coconut water (CW) on callus and protocorm-like body (PLB) induction from root tips of Oncidium varicosum grown in vitro were studied. Relatively high concentrations of NAA favoured callus induction. PLBs were obtained from callus culture either in medium with 15% CW+1.25 mg/1 NAA or in the presence of CW alone. The low regeneration capac ity of buds presented by the calli seems to be the result of an incomplete dedifferentiation of their cells. This seems to keep the callus in a root state. The apparent presence of high cytokinin levels either in the explant or in the callus may be related to these processes.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid     

High level extracellular secretion of thermostable α-amylase ofBacillus stearothermophilus inEscherichia coli

Summary Bacillus stearothermophilus BR135 (ATCC 29609)amy gene was cloned in pBR322 from its plasmid DNA and was subcloned in a vector useful both forB. subtilis andE. coli.E.coli HB101 harboring the plasmid pSS099 when grown in L medium in presence of 5. g/ml chloramphenicol produces 70 units/ml of extracellular -amylase. This is nearly twice that ofE.coli cells harboring pSSO76, a plasmid havingamy ofB.stearothermophilus BR135 atHindIII site of pBR322. Characteristically the protein was a 58 kd protein and cross reacted with antiserum developed against purified -amylase of BR135.     

Interspecific somatic hybrids ofRudbeckia hirta andR. Laciniata (Compositae)

Interspecific somatic hybrid plants betweenRudbeckia hirta cv. Marmalade andR.laciniata cv. Irish Eyes were regenerated following the electro-fusion of mesophyll protoplasts ofR.hirta with callus protoplasts ofR.laciniata. A hybrid selection scheme was based on the fact that plant regeneration, from parental protoplasts ofR.hirta, was via shoot regeneration of callus, and only via rhizogenesis forR.laciniata. The other half of the selection strategy was based on the presence of anthocyanin-pigmented roots; a characteristic of theR.hirta parent only. Somatic hybrids were regenerated, via rhizogenesis, alongside normalR.laciniata but were distinguished by the presence of pigmented roots (a feature ofR.hirta). Hybrid plants had a floral morphology that was intermediate as compared to that of the two parents, with an expected somatic chromosome number of 2n=(2x+4x)=74. Pollen viability though was low. Esterase and peroxidase isozyme profiles confirmed the hybrid nature of the regenerated plants with pigmented roots, whilst chloroplast DNA restriction analysis showed that these hybrids had aR.laciniata chloroplast DNA. This demonstration of somatic hybridisation not only opens up the possibility of incorporating novel traits between such ornamentalCompositae species, but provides a selection strategy based on rhizogenesis as the route to plant regeneration coupled with heritable pigmentation production of roots as a confirmatory hybrid marker.ABBREVIATIONS BSA bovine serum albumin - EDTA ethylene diamine tetra acetic acid - FDA fluorescein diacetate - f.wt. fresh weight - IAA indole 3-acetic acid - MS Murashige and Skoog (1962) medium - TEMED N,N,N,N-Tetra methyl ethylene diamine - TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid)     

Production of theanine and other γ-glutamyl derivatives by Camellia sinensis cultured cells

The maximum theanine production by Camellia sinensis cultured cells was achieved by culturing in the modified MS medium containing 2 mg/l indole-3-butyric acid, 0.1 mg/l kinetin, 0 mM NH₄NO₃ and 39.6 mM KNO₃ with 40 mM ethylamine hydrochloride or 20 mM ethylamine hydrochloride and 10 mM L-glutamic acid. Other primary amines, such as methylamine, n-butylamine, 2-hydroxyethylamine, 2cyanoethylamine, aniline, benzylamine and phenylethylamine, were also biotransformed to N⁵-alkyl-L-glutamine derivatives by C. sinensis cultured cells.Abbreviations IBA indole-3-butyric acid - K kinetin - MS medium Murashige and Skoog's basal medium (1962) - NMR nuclear magnetic resonance Part 70 in the series Studies on Plant Tissue Culture. For Part 69 see Furuya et al. (1990).     

Secondary product formation by cultures of Beta vulgaris and Nicotiana rustica transformed with Agrobacterium rhizogenes

Hairy root cultures of Beta vulgaris and Nicotiana rustica were established after roots were induced on plants following infection with Agrobacterium rhizogenes. The transformed cultures of B. vulgaris and N. rustica synthesised their characteristic secondary products, the betalain pigments and nicotine alkaloids respectively, at levels comparable with those of in vivo roots from the same variety. Betalains were entirely retained inside the root tissue. In contrast, a proportion of the nicotine alkaloids was secreted into the medium. The potential of this type of in vitro plant tissue culture for the production of valuable plant secondary products is identified and confirmed.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, -naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2–1 M) of -naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2 ⁺ protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of -naphthalene acetamide (between 30 and 300 M) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2 ⁺ and gene 2 ^– cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.     

Plant regeneration from cultured hypocotyl explants of diploid and tetraploid Solanum khasianum clarke

Plant regeneration through shoot bud formation was induced in cultured hypocotyl explants of diploid and tetraploid varieties of Solanum khasianum. Optimum regeneration of multiple shoot buds accompanied by rooting occurred on Murashige and Skoog's medium supplemented with IAA (1 mg/1) and kinetin or benzyladenine (1 mg/1). Plantlets obtained in vitro were successfully established in soil.Part of a thesis entitled Morphogenetic studies in tissue and organ cultures of certain Solanaceous plants: Capsicum, Lycopersicon and Solanum submitted by A.L. Gunay and approved for Master's degree in Botany by research of the University of Bombay.     

Xylanase and β-xylosidase production byAspergillus niger NCIM 1207

Summary Aspergillus niger NCIM 1207 producing significantly high levels of -glucosidase was found to secrete hemicellulolytic enzymes (xylanase and -xylosidase) in the culture medium. High yields of -xylosidase were obtained when it was grown on either xylan (3%) or wheat bran (4%). Cellulose was a poor inducer of -xylosidase. The pH and temperature optima for-xylosidase were 4.5 and 65°C respectively.NCL Communication No. 3751