Shoot regeneration from mesophyll protoplasts and leaf explants of Rehmannia glutinosa

Mesophyll protoplasts obtained from leaves of shoot cultures of Rehmannia glutinosa were cultured in Murashige and Skoog (1962) liquid or liquid-over-agar medium containing 2.0 mg L^?1 naphthaleneacetic acid and 0.5 mg L^?1 benzylamino purine. An amino acid mixture of glutamine, arginine, glycine, and aspartic acid promoted sustained protoplast division, with an average plating efficiency of 27%. Protoplast-derived colonies formed callus which readily regenerated shoots on fransfer to Murashige and Skoog based agar medium with 2.0 mg L^?1 indoleacetic acid and 1.0 mg L^?1 benzylamino purine. Leaf explants also showed a marked capacity for shoot regeneration in culture.     

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 μm and 10 μm). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 μm) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 μm) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.     

<Emphasis Type="Italic">Cytisus aeolicus</Emphasis> Guss. ex Lindl. <Emphasis Type="Italic">in vitro</Emphasis> cultures and genistin production

Cytisus aeolicus Guss. ex Lindl. (Fabaceae family, subfamily Faboideae) is an endangered endemic species of the Aeolian Islands, Sicily. In vitro multiplication of C. aeolicus shoots was described in this work and cell cultures were established from cotyledons and hypocotyls to investigate their potential production of isoflavones. Aseptically germinated seeds, cultivated on LS modified basal medium, gave the initial explants used both to induce axillary propagation and callus cultures. The LS (Linsmaier and Skoog) basal medium, supplemented with 0.1 mg L^?1 of 6-benzylaminopurine were used to induce axillary propagation. The callus induction was performed using the basal medium added with 5 mg L^?1 2,4-dichlorophenoxy acetic acid and 5 mg L^?1 kinetin (control medium). Basal medium was also added with 2000 mg L^?1 casein hydrolysate (CH) or 900 mg L^?1myo-inositol (MI). C. aeolicus callus cultures on CH and MI media produced an unique compound, the isoflavone genistein 7-O-ß-D-glucopyranoside (genistin), which has not previously been isolated from wild plants. Callus cultures grown on the medium containing myo-inositol produced the greatest amount of genistin. C. aeolicus tissue culture procedures could provide suitable plant material both for germplasm preservation (by micropropagation) and for biotechnological selective isoflavone production (by callus culture).     

Auxin-facilitated utilization of glutamic acid by tobacco crown-gall teratoma cells

Summary Teratoma tissues obtained by inoculating Nicotiana tabacum cv. Turkish with a moderately virulent strain of the crown-gall bacterium require the synthetic auxin, -naphthaleneacetic acid (NAA) when glutamic acid is used as a sole nitrogen source in the culture medium. In contrast, growth on culture media containing ammonium ion, nitrate ion or glutamine as an N source does not require NAA. Moreover, added NAA does not stimulate teratoma tissue grown on these N sources. Glutamic acid did not inhibit growth of teratoma tissue on media containing NO ₃ ^- . Growth on mixtures of glutamic acid and NO ₃ ^- was additive in the presence of NAA indicating that NAA promotes the utilization of glutamic acid in the culture medium. Increased concentration of potassium ion in the culture medium was required for growth on glutamic acid in the absence of added auxin. K⁺ did not stimulate growth on glutamine. When teratoma tissues were grown on media containing glutamic acid and varying concentrations of both K⁺ and NAA increasing concentrations of NAA reduced the stimulating effect of K⁺ and, conversely, increasing concentrations of K⁺ reduced the stimulating effect of NAA. It is concluded that K⁺ and auxin act either directly or indirectly at a common site to promote glutamic acid utilization.     

Establishment of long-term proliferating shoot cultures of elite <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l^?1 benzyladenine (BA) and 0.1 mg l^?1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l^?1 BA along with 1.0 mg l^?1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l^?1 thidiazurone (TDZ) and 0.1 mg l^?1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l^?1 BA and 0.1 mg l^?1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Cherry fruit development: The use of a microassay for studying indoleacetic acid oxidase

An improved procedure is described for extraction and assay of indoleacetic acid oxidase from seeds of sour cherry (Prunus cerasus L.). The extraction procedure was optimized for pH, buffer, polyvinylpolypyrrolidone (PVP) and tissue: buffer ratio. Greatest extraction efficiency was obtained at pH 4.0, 0.2 M acetate buffer, tissue: PVP ratio of 1:2.5 and tissue: buffer ratio of 50 ml per g of seed. The enzyme was assayed at 30°C using indoleacetic acid-1-¹⁴C as substrate and radioassaying the ¹⁴CO₂ evolved. Mn²⁺ and 2,4-dichlorophenol enhanced enzyme activity but were not obligatory. A minimum substrate concentration of 60 M was needed for quantitative evaluation. This assay was sensitive and reproducible, enzyme activity being demonstrated in as little as 0.8 mg of seed tissue with a coefficient of variation of 1 to 9%.     

Some effects of plant growth regulators on tissue cultures of the marine red alga Agardhiella subulata (Gigartinales,Rhodophyta)

We examined whether auxins and cytokinins, either singly or in combination, stimulate cell division in tissue cultures of a red seaweed. Our experimental model consisted of filamentous and callus-like growths that developed from cross-sectional discs cut from young branches of Agardhiella subulata. Plant growth regulators were added to the medium to give combinations of an auxin with a cytokinin over a range of concentrations (1 µg L^–1 –10 mg L^–1). Several mixtures of auxins and cytokinins, as well as some single auxins, cytokinins and phenolics, stimulated cell division and growth in the tissue cultures beyond that of controls. The treatments that were effective included: phenylacetic acid/zeatin; phenylacetic acid/6-benzylaminopurine; -naphthaleneacetic acid/zeatin; 2,4,5-trichlorophenoxyacetic acid/6-benzylaminopurine; and indoleacetic acid/kinetin. High concentrations of cytokinins (i.e. 10 mg L^–1) inhibited the regeneration of plants in some of the cell cultures. These results provide further evidence that growth regulators can be used for the tissue culture of seaweeds and for the study of developmental phenomena in these plants.     

Preparation and characterization of the tissue culture of spruce (Picea excelsa Link)

The paper describes isolation and partial characterization of the tissue culture of the common spruce (Picea excelsa Link). The tissue culture was grown on the medium ofHarvey (1967) modified by increasing the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) to l mg l^?1 and by omitting the tyrosine. By selective 10-month subculturing the tissue culture was obtained possessing relatively stable properties and giving good yields of biomass. The tissue of the callus was of yellowish colour, compact and homogenous. The obtained tissue culture was characterized by its growth curve and by following the O₂ consumption during the growth.     

Culture medium influence on growth,fatty acid,and pigment composition of <Emphasis Type="Italic">Choricystis minor</Emphasis> var. <Emphasis Type="Italic">minor</Emphasis>: a suitable microalga for biodiesel production

The purposes of this study were to assess the influence of culture medium on biomass production, fatty acid, and pigment composition of Choricystis minor var. minor and to evaluate the use of this microalga as a source of fatty raw material for biodiesel production. Cultures of C. minor var. minor were grown using WC (Wright’s cryptophyte) and BBM (Bold’s Basal medium) media. BBM medium produced more biomass (984.3 mg L^?1) compared to the WC medium (525.7 mg L^?1). Despite this result, WC medium produced a higher methyl ester yield for biodiesel production than the BBM medium (170.0 and 90.2 mg g^?1 of biomass, respectively). The average percentage of fatty acids obtained using the WC medium (17.0 %) was similar to soybean (18.0 %) and with similar biomass fatty acid profile. However, for pigment production, carotenoids and chlorophyll concentrations were twice as high when using the BBM medium.     

Free tryptophan and indole-3-acetic acid levels in the leaves and vascular pathways of Ricinus communis L.

Levels of free tryptophan in the leaves, phloem and xylem saps of Ricinus communis L. were determined by colorimetric assay. Values of 0.38 g ml^-1 in root pressure sap and 96.0 g ml^-1 in phloem sap were recorded. Tryptophan levels were highest in mature and senescing leaves. Levels of indoleacetic acid (IAA) in the phloem sap and leaves were determined by gas chromatography—mass spectrometry using a deuterated internal standard. A mean value of 13.0 ng ml^-1 was recorded in phloem sap. The distribution in the leaves showed an inverse relationship to that of tryptophan, being highest in young leaves.Abbreviations IAA indoleacetic acid - GC-MS Gas chromatography-mass spectrometry - PFP-derivative pentafluoropropionyl-derivative - TLC thin layer chromatography     

Neutral amino acid transport in an androgen-dependent epithelium

• 1.1. Transport of neutral amino acids by the isolated seminal vesicle epithelium of normal and gonadectomized guinea pigs has been investigated by measurement of the uptake of 2-amino[1-¹⁴C]-isobutyric acid and 2-methylamino[1-¹⁴C]isobutyric acid.
• 2.2. The V_max for Na-dependent and -independent transport of both amino acids was reduced by gonadectomy but the general transport characteristics appeared to be unchanged by this treatment.
• 3.3. The most likely explanation of the decreased transport is the loss of transporter molecules associated with the tissue regression that follows rapidly on gonadectomy.

     

An improved method for callus proliferation and regeneration of Fuchsia hybrida

Best callus initiation was obtained when single-node explants of Fuchsia hybrida were incubated in the light on Gamborg B5 medium containing 5×10^-6 M indoleacetic acid and benzylaminopurine at 5×10^-7 M or 10^-6 M. Healthy callus proliferation was maintained in darkness on full-strength B5 medium supplemented with 5×10^-6 M IAA and 5×10^-7 M BAP. Regeneration from callus was obtained in 3 to 6 weeks, using half-strength hormone-free Campbell & Durzan medium.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - SE standard error     

Influence of culture conditions on embryo formation and maturation in auxin-induced embryogenic cultures of Digitalis obscura

Somatic embryogenesis was induced in hypocotyls of Digitalis obscura using indoleacetic acid or 2,4-dichlorophenoxyacetic acid with different culture and subculture conditions. Indoleacetic acid-induced embryogenic cultures were used to investigate the effects of amino acids, polyamines and growth regulators on embryo differentiation and maturation. Supplementation of the media with amino acids, polyamines or abscisic acid did not influence or had an adverse effect on embryogenic response. Gibberellic acid at 1.4 M in either culture (30 days) or subculture medium was effective in promoting both differentiation and normal embryo development. The efficiency of somatic embryogenesis was greatly enhanced when isolated indoleacetic acid-induced proembryogenic masses were subcultured in liquid medium with reduced auxin content.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - IAA indoleacetic acid - Ptr putrescine - Spd spermidine - Spn spermine     

Isolation and Identification of O-Acetyl and 12-Hydroxyradiclonic Acids

An N-acetylglutamate-acetylornithine acetyltransferase-deficient arginine-requiring mutant AA–1, was derived from an l-arginine producer of Corynebacterium glutamicum. It accumulated a large amount (30 mg per ml) of l-glutamic acid and a small amount (1.2 mg per ml) of N^α-acetylornithine, an intermediate of arginine biosynthesis, in the culture medium.

The production of N^α-acetylornithine by AA–1 was not affected by the concentration of l-arginine in the medium, whereas that of l-glutamic acid was inhibited by a high concentration of l-arginine in the medium containing excess biotin.     

Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with -naphthaleneacetic acid (NAA) (1.0 mg 1^-1) and benzylamine purine (BA) (0.5 mg 1^-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1^–1), BA (0.5 mg 1^–1) and casein hyrdrolyzate (2.0 mg 1^-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.MS at full strength was found inhibitory for callus induction and proliferation, but 1/2 MS was suitable. Similarly callus growth was very slow at 25°C but increased when the temperature was lowered to 15°C as did bud initiation.Abbreviations BA benzylamine purine - IAA indoleacetic acid - K kinetin (6-furfurylamino purine) - NAA -naphthalene acetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Clonal propagation of mature elite trees of Commiphora wightii

Elite trees of Commiphora wightii (Arnott) Bhandari were selected from the wild on the basis of their content of guggul, an oleoresin. The selected tree was micropropagated through forced axillary branching on Murashige and Skoog's (MS) medium supplemented with benzyladenine (BA) and kinetin. Highest frequency of shoot formation was achieved on MS medium supplemented with 17.8 M BA, 18.6 M kinetin, 100 mg l^-1 glutamine, 10 mg l^-1 thiamine HCL and 0.3% activated charcoal. Seasonal changes affected the shoot proliferating potential of the initial explants in vitro. Transfer of shoots to a medium containing a lower concentration of BA (1.8 M) and kinetin (1.9 M) before rooting markedly stimulated shoot elongation. Shoots could be rooted by treating them with both indoleacetic acid and indolebutryic acid for 24 h in darkness and transferring them to a low-salt basal medium with activated charcoal. After rooting, transfer to a half-strength White's (modified) medium was necessary for further development of the plantlet. Regenerated plantlets were successfully established in soil.Abbreviations AC activated charcoal - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - 2-iP isopentenyladenine - MS Murashige and Skoog's medium - NAA -naphthaleneacetic acid - PG phloroglucinol     

Regeneration of mortiño (<Emphasis Type="Italic">Vaccinium floribundum</Emphasis> Kunth) plants through axillary bud culture

The use of conventional propagation strategies for Vaccinium floribundum Kunth has proven difficult, which has resulted in this species’ escape from formal cultivation, despite its importance as a gastronomic and nutraceutical fruit. The current report presents an efficient propagation methodology for V. floribundum using axillary bud growth. Axillary buds were cultured on modified Woody Plant medium (mWPM) supplemented with 3.0 mg L^?1 N6-isopentenyladenine (2iP) or with 5.0 mg L^?1 2iP plus 0.1 mg L^?1 1-naphthaleneacetic acid (NAA). The best results for plant propagation were obtained on mWPM with 2iP and NAA, where significantly higher numbers of shoots per bud were observed. In vitro-rooted plants were successfully acclimatized to a peat and vermiculite substrate, while unrooted plants could be efficiently grown after an ex vitro rooting treatment by submersion in an 0.5 g L^?1 indole-3-butyric acid (IBA) or potassium IBA (KIBA) solution. This is the first report of an efficient propagation methodology for V. floribundum using plant tissue culture protocols, and provides a tool for the implementation of conservation strategies for this species.