Immunodetection and Identification of N-(o-Hydroxybenzylamino)Purine as a Naturally Occurring Cytokinin in Populus x canadensis Moench cv Robusta Leaves

A highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for 9-β-d-ribofuranosyl-N⁶-(o-hydroxybenzylamino)purine [(oOH)[9R]BAP] and structurally related cytokinins. As little as 3 femtomoles of the compound could be detected by this method. Cross-reactivity studies demonstrated the specificity of four polyclonal antibodies for (oOH)[9R] BAP and its free base in preference to a range of natural cytokinins and other purines. After evaluating the method by internal standardization employing [2-³H](oOH)[9R]BAP of high specific radioactivity as recovery marker by dilution analyses and by immunohistograms, it was possible to apply ELISA to quantify (oOH)[9R]BAP in plant extracts. In addition to (oOH)[9R]BAP, an unknown cytokinin reacting with the same antibody was detected in high performance liquid chromatography-fractionated extracts of mature Populus × canadensis Moench cv Robusta. The structure of the new compound was determined by gas chromatography-mass spectrometry and finally confirmed by synthesis as N⁶-(o-hydroxybenzylamino)purine.     

Seasonal changes in sugar beet photosynthesis as affected by exogenous cytokininN 6-(m- hydroxybenzyl)adenosine

Foliar sprays withN ⁶-(m-hydroxybenzytyadenosine, (mOH)-[9R]BAP, one of the synthetic cytokinins, were applied to field-grown sugar beet twice during the vegetation period: before full canopy closing(I), 6 weeks before harvest(II) or both. Application of (mOH)[9R]BAP retained high cytokinin content that usually declines prior to harvest. The total content of isoprenoid cytokinins at harvest was 2.6-fold higher in (mOH)[9R]BAP-treated plants as compared to the controls. TreatmentI had no significant effect on contents of chlorophylls (Chl)a andb and carotenoids, nor on the rates of net photosynthesis (P_N) or photorespiration (R_l), or on CO₂ compensation concentration (Γ) measured during the whole vegetation period on detached leaves under optimum environmental conditions. Increased values in P_N, R_L and Chla andb contents were found in variantsII and I+II linked with a delay in leaf senescence before harvest. Transpiration rate and stomatal conductances of adaxial and abaxial epidermes were not significantly affected by any treatment.     

Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N⁶-(²-isopentenyl)adenine ([2,8-³H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent K_m value of the enzyme was 4.3 M for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-³H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl₂) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent K_m value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.Abbreviations Ade adenine - iP N⁶-(²-isopentenyl)adenine - [2,8-³H]iP [2,8-³H]-N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - (diH)iP N⁶-isopentyladenine - (diH)Z dihydrozeatin - BAP N⁶-benzyladenine - ( _o OH)[9R]BAP N⁶-(o-hydroxybenzyl)adenosine - (mOH)[9R]BAP N⁶-(m-hydroxybenzyl)adenosine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthalene-1-acetic acid - ABA abscisic acid - Con A concanavalin A     

Benzyladenine and derivatives-their significance and interconversion in plants

Recently benzyladenine has been isolated as a natural cytokinin from a number of plants. The natural occurrence of this cytokinin will change the attitude with which physiologists view this hormone. This review attempts to put into context what is known about this cytokinin and its derivatives and to compare and contrast its metabolism and the function and physiological action of its various metabolites. Nothing is known about the biosynthesis of benzyladenine. Its structure would suggest that its biosynthetic pathway may differ considerably from that of zeatin and iso-pentenyladenine.Abbreviations Ade adenine - Ado adenosine - BA benzyladenine - [9R]BA BA ribonucleoside - [9R-MP]BA BA nucleotide - [9R-DP]BA BA dinucleotide - [9R-TP]BA BA trinucleotide - [3G]BA BA 3 glucoside - [7G]BA BA 7 glucoside - [9G]BA BA 9 glucoside - [9R-G]BA BA 9-ribosylglucoside - [9Ala]BA BA alanine-conjugate - (2OH)BA BA ortho-OH - (2OH)[9R]BA BA ortho-Oh-riboside - KN kinetin - [9R]KN KN ribonucleoside - DHZ dihydrozeatin - Z trans-zeatin - [9R]Z zeatin ribonucleoside - [7G]Z zeatin-7-glucoside - [9G]Z zeatin-9-glucoside - [9Ala]Z zeatin alanine-conjugate - (OG)[9R]Z O-glucoside of zeatin ribonucleoside - [9R-MP]Z zeatin nucleotide - iP iso-pentenyladenine - [9R]iP iP ribonucleoside     

Effects of aluminium on growth and root reactions of phosphorus stressed Betula pendula seedlings

The impact of two constant non-toxic levels of Al stress (0.2 and 0.4 mM) on growth and ³²P uptake capacity on sub-optimal (P-limited) Betula pendula seedlings grown in sand culture was examined.Seedling growth was under optimum controlled conditions in a growth chamber where nutrient additions were made at a predetermined relative addition rate (R_A) of 10% day^-1. Three treatment groups of seedlings 0, 0.2 and 0.4 mM Al were harvested at 15, 29 and 42 days. The excised roots were exposed to a ³²P-labelled solution for 15 minutes to measure their capacity for P uptake. Growth was determined by weighing the roots, stems and leaves of the seedlings.Growth data showed that relative growth rate (R_G) should equal the R_A of P the most limiting nutrient, which was supplied at P/N 3% instead of an optimal 15%. Therefore, Ingestad's theory can also be used succesfully in sand culture and this may be particularly important for future studies of root and rhizosphere exudates. Low levels of Al (< 0.2 mM) in combination with low P supply significantly lowered the R_G of the birch seedlings by further reducing P supply. However, previous studies of birch seedling growth and nutrient uptake using Ingestad's solution culture technique with optimumal P supply did not show any effect of Al on growth untill the Al was in excess of 3 mM. Aluminium was not directly toxic to the plants and therefore roots could respond to the ³²P bioassay.     

Effects of silicon on enzyme activity and sodium,potassium and calcium concentration in barley under salt stress

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown in a hydroponics system containing 120 mol m^-3 NaCl only and 120 mol m^-3 NaCl with 1.0 mol m^-3 Si (as potassium silicate). Compared with the plants treated with salt alone, superoxide dismutase (SOD) activity in plant leaves and H⁺-ATPase activity in plant roots increased, and malondialdehyde (MDA) concentration in plant leaves decreased significantly for both cultivars when treated with salt and Si. The addition of Si was also found to reduce sodium but increase potassium concentrations in shoots and roots of salt-stressed barley. Sodium uptake and transport into shoots from roots was greatly inhibited by added Si under salt stress conditions. However, Si addition exhibited little effect on calcium concentrations in shoots of salt-stressed barley. Thus, Si-enhanced salt tolerance is attributed to selective uptake and transport of potassium and sodium by plants. The results of the present study suggest that Si is involved in the metabolic or physiological changes in plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of 6-Benzylaminopurine on the Starch Metabolism of Stellaria media

The effect of 6-benzylaminopurine (BAP) was studied by measuring the amylase activity of leaf extracts and the starch content of chloroplasts in Stellaria media-plants cultivated for five days in solutions containing different quantities of BAP. Small amounts of BAP decreased amylase activity in weak-growing plants. In strong-growing ones the effect was variable. However, in all experiments very high amount of this growth regulator caused a great increase in amylase activity which was accompanied by a relatively high concentration of starch in the chloroplasts. The induction of amylase activity was thus not high enough to diminish the starch content. This is supposed to be due to the lack of a sink organ since the growth of roots and shoots was greatly inhibited in concentrated BAP-solutions (10^-5M or more). In these cases the plants usually died during the first week. In some cases small amounts of BAP increased the growth of S. media, and high concentrations inhibited it causing characteristic morphological changes, such as creeping growth pattern, small, thick leaves and the turning up of the leaf margins. Benzylaminopurine also caused increased resistance to aging, especially in low concentrations (10^–7–10^–6M).     

Distribution and Partitioning of Newly Taken-up Boron in Sunflower

Distribution and partitioning of newly acquired boron (B) in a mature sunflower (Helianthus annuus L., cv. 3101) plant was investigated. In leaf blades of sunflower plants grown under 0.93, 2.8, and 9.3 mmol B m⁻³, the level of cell-wall-bound B was rather uniform, irrespective of leaf position and B concentration. Boron concentration gradients among leaf positions were produced mainly by different levels of water-soluble B. To determine the distribution of newly taken-up B in plant parts, ¹⁰B-labeled boric acid at a concentration of 2.8 mmol B m⁻³ was applied. The majority of newly acquired B was delivered to the younger leaves, however, approximately one-fourth of the B in the top and second leaves was the older B which was taken up before the 6 d treatment period. In the root tissues, two-fifth of the water-soluble B was new B taken up in the last 6 d, however, within 6 h of the application new B contributed to approximately 80% of the xylem sap B, suggesting that newly taken-up B is preferentially transported to the shoots. When B was withdrawn from the culture solution, the B concentration per leaf area of the lower leaves decreased slightly over 9 d. However, there was an abrupt decrease in the younger leaves, even when taking into account the rapid expansion of the leaf blade, suggesting that B moves more rapidly from the younger leaves than from the older leaves     

HCO−3 uptake through the roots and its effect on the productivity of willow cuttings

Abstract Young willow plants (Salix‘aquatica gigantea’) were grown in hydroponic culture media, and ¹⁴C–labelled sodium bicarbonate was fed to the roots. Uptake of ¹⁴C-label in the leaves and shoots was assayed after two different feeding periods (6 h, 48 h). Even during the shortest feeding period, ¹⁴C-label had been transferred to the leaves and shoots. Compared with the longer feeding period, after the 6 h feeding period more label was in the form of acid-labile products, whereas after the 48 h feeding period most of the label was in acid-stable products. A second experiment was designed to test whether carbon uptake by roots affects the growth of young willow plants. Uniform rooted cuttings were grown in hydroponic cultures at five different levels of bicarbonate: 0, 0.015, 0.147 0.737, and 1.473 mol m^?3 NaHCO₃. After a 4-week growing period we determined the biomass of leaves, shoots, roots and cuttings. Production of total dry matter (shoots, leaves and roots) increased with increasing bicarbonate concentration. Saturation of dry matter production was reached at 0.737 mol m^?3 NaHCO₃, but a higher concentration of NaHCO₃ (1.470 mol m^?3) caused a slight decrease in the dry matter production. At 0.737 mol m^?3 NaHCO₃ the total dry weight increased by 31.1%, which suggests that uptake of dissolved carbon dioxide through the roots might affect carbon budgeting in young willow plants.     

Time-course of Nitrate Uptake and Nitrate Reductase Activity in Nitrogen-depleted Dwarf Bean

The rate of nitrate uptake by N-depleted French dwarf bean (Phaseolus vulgaris L. cv. Witte Krombek) increased steadily during the first 6 h after addition of NO₃ -After this initial phase the rale remained constant for many hours. Detached root systems showed the same time-course of uptake as roots of intact plants. In vivo nitrate reductase activity (NRA) was assayed with or without exogenous NO₃^- in the incubation medium and the result ing activities were denoted potential and actual level, respectively. In roots the difference between actual and potential NRA disappeared within 15 min after addition of nitrate, and NRA increased for about 15 h. Both potential and actual NRA were initially very low. In leaves, however, potential NRA was initially very high and was not affected by ambient nitrate (0.1–5 mol m^-3) for about 10 h. Actual and potential leaf NRA became equal after the same period of time. In the course of nitrate nutrition, the two nitrate reductase activities in leaves were differentially inhibited by cycloheximide (3.6 mmol m^-3) and tungstate (1 mol m^-3). We suggest that initial potential NRA reflects the activity of pre-existing enzyme, whereas actual NRA depends on enzyme assembly during NO₃^- supply. Apparent induction of nitrate uptake and most (85%) of the actual in vivo NRA occurred in the root system during the first 6 h of nitrate utilization by dwarf bean.     

Involvement of cytokinin in the stimulation of nodulation by low concentrations of ammonium in Pisum sativum

Nodulation in pea (Pisum sativum L.) grown in hydroponic and sand culture systems is stimulated by low concentrations (<1.0 mM) of ammonium, but the physiological mechanisms underlying this stimulation are unknown. The current study involves a series of experiments, which investigate if the ammonium‐induced stimulation of nodulation involves changes in endogenous hormone (auxin and cytokinin) levels. P. sativum L. cv. Express was grown in growth pouches for 1 week with mineral N (0.5 and 2.0 mM NH₄⁺ or NO₃^–) or for 3 weeks exposed to exogenous indole‐3‐acetic acid (IAA) or 6‐benzylaminopurine (BAP) at a range of concentrations (10^‐9?10^‐5 M). Ammonium enhanced nodulation on the basis of both early whole plant (nodules plant^?1) and specific nodulation (nodules g^?1 root DW), especially in 0.5 mM treatment in which nodulation was approximately 4‐fold of the mineral‐N‐free control 1 week after inoculation. Correspondingly, the roots treated with ammonium contained much higher levels of t‐zeatin (Z) and lower t‐zeatin riboside (ZR) than that the control or nitrate‐treated plants. There was no significant difference in IAA levels between the control and ammonium treatments. Exogenous application of BAP for 3 weeks at concentrations of 10^‐7?10^‐5 M strongly inhibited nodulation. However, 10^?9 M BAP, but not IAA, significantly enhanced nodulation. These data support the theory that a relatively high ratio of cytokinin:auxin in roots is favourable for nodule initiation, but that an excessively high level of cytokinin inhibits nodulation. Based on these results we propose that stimulation of nodulation by low concentrations of ammonium may be mediated through increasing Z level in roots, which alters the balance of cytokinin and auxin, which in turn induces cortical cell divisions leading to nodule initiation.     

Significance of organic nitrogen acquisition for dominant plant species in an alpine meadow on the Tibet plateau, China

Though the potential of plants to take up organic N (e.g., amino acids) is well established, the true significance of organic N acquisition to plant N nutrition has not yet been quantified under field conditions. Here we demonstrate that organic N contributes significantly to the annual N uptake of three dominant plant species (Kobresia humilis, Saussurea superba and Stipa aliena) of alpine meadows on the Tibet Plateau, China. This was achieved by using double-labelled (¹⁴C and ¹⁵N) algae as a source for slow and continuous release of amino acids, and tracing both labels in the above- and below-ground plant biomass. Four months after addition of algae, between 0.5% and 2.6% of ¹⁴C and 5% and 14% of ¹⁵N from added algae were recovered in the plants, which translate into an uptake of organic N between 0.3 mg N m⁻² and 1.5 mg N m⁻². The calculated contribution of organic N to total N uptake was estimated to range between 21% and 35% for K. humilis, and between 13% and 21% for S. aliena and S. superba, respectively, implying that organic N uptake by grassland plants is quantitatively significant under field conditions in the studied alpine meadows. This finding has important ecological implications with regard to competition for organic N between microorganisms and plant roots.     

Mobilization of the reserve N in citrus

The mobilization of N from reserve organs (leaves, roots, branches and trunk) to developing new organs was studied at different moments of the growth cycle. Three-year-old Valencia Late orange trees (Citrus sinensis (L.) Osbeck) were grown individually outdoors in 150 L containers filled with siliceous sand. Trees were irrigated with a nutrient solution labelled with potassium nitrate with an enrichment of 4 atom ¹⁵N % excess during a complete growth cycle. At the following year, plants were irrigated with unlabelled nutrient solution, and harvested throughout the growth cycle (flowering, fruit set, second flush, third flush, and dormancy). Total N and ¹⁵N analyses were carried out in the different organs of the plants.The highest amounts of N were found in leaves and roots (33–42% and 30–38%, respectively). Distribution of ¹⁵N was similar to that obtained for total N (42 and 39% of total ¹⁵N in leaves and roots, respectively) as is expected after a long period of labelling. Old leaves were the main reserve organs, contributing a 40–50% of the total N exported. Roots and aerial woody tissues exported between 30–35% and 15–25% of total reserve N, respectively. N exported by old leaves was approximately 57% of the N accumulated during the preceding year, while roots translocated 40% and trunk plus branches 35%. More than 70% of N accumulated in new organs during spring came from N stored in old organs.     

Growth responses and Hyoscyamine content ofDatura innoxia under the influence of coal-smoke pollution

This study evaluated the impact of pollution on growth responses inDatura innoxia. Coal-smoke emissions were produced by the Badarpur Thermal Power Plant in Delhi, India. At the polluted site, the size of roots and leaves as well as the number of branches and leaves per plant increased, but shoot lengths and leaf areas were lower, compared with control plants. The net photosynthesis rate, stomatal resistance, and the amount of pigments (chlorophyll a, b, and carotenoids) were less in pollution-affected plants, while stomatal conductance and intercellular CO₂ concentration were higher in these plants. Explants from both sites (polluted and non-polluted), grown in vitro on various combinations of auxin (2,4-D, NAA) and cytokinin (BAP, KN), showed the maximum response on a medium containing NAA (0.1 mg L^-1) with BAP (5.0 mg L^-1). Hyoscyamine content was higher in all parts (root, stem, leaf, and regener-ants) of the polluted plants.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.     

Efficient Synthesis of [2-15N]Guanosine and 2′-Deoxy[2′-15N]Guanosine Derivatives Using N-(tert-Butyldimethylsilyl)[15N]Phthalimide as a 15N-Labeling Reagent

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-6-chloro-2-fluoro-9 H-purine with N-(tert-butyldimethylsilyl)[ ¹⁵ N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[ ¹⁵ N]phthalimidopurine derivative, which was subsequently converted to the [2-¹⁵N]guanosine derivative was also efficiently synthesized through a similar procedure.     

Establishment of <i>Rhodiola quadrifida</i> Hairy Roots and Callus Culture to Produce Bioactive Compounds

Rhodiola quadrifida is a rare mountain medicinal plant whose root extracts are used in traditional Chinese medicine as a hemostatic, antitussive, and tonic in the treatment of gynecological diseases. The aim of the study was to obtain R. quadrifida cultures at different degrees of differentiation in vitro and compare their growth characteristics and the content of salidroside and rosavin. Hairy roots were obtained by incubating cotyledons and hypocotyls in a suspension of Agrobacterium rhizogenes strain A4. The presence of the rolB and rolC genes was proven by polymerase chain reaction. The obtained roots were cultivated in Murashige-Skoog medium (MS). Calluses were obtained from the hairy roots in MS medium with the addition of hormones: 3 mg/L 2,4 D and 0.5 mg/L BAP. The presence of the main secondary metabolites of R. quadrifida, salidroside and rosavin, in calluses and salidroside in hairy roots by HPLC/MS was confirmed. The content of salidroside in callus culture was significantly higher than in hairy roots, 0.158 and 0.047%, respectively. The content of rosavin in callus culture was 0.07%. The content of rosavin and salidroside in callus culture was close to the level of these substances in the rhizomes of R. quadrifida plants growing in vivo, making this culture promising for its possible biotechnological use.     

Effects of NO3– and BAP on organogenesis in tissue-cultured shoot primordia induced from shoot apices of Utricularia praelonga St. Hil.

The effects of NO₃ ^– and BAP on organogenesis in shoot primordia of Utricularia praelonga subcultured in B5 liquid medium were studied. In B5 liquid basal medium supplemented with 24.73 mM KNO₃ and 2.0 mg/l BAP the subcultured shoot primordia continuously multiplied into numerous small, globular masses, while with dilution of the KNO₃ to 3 mM organogenesis was promoted. Pulse treatment of the shoot primordia with 3 mM KNO₃ in B5 liquid medium for 72 h and then transplantation to the B5 basal liquid-medium induced meristemoids in this tissue. When the shoot primordia regenerated meristemoids, they never reverted back into the proliferation cycle. The addition of BAP in the B5 liquid medium with 3 mM KNO₃ regulated the differentiation rate of the stems and leaves in the meristemoids induced in the masses of shoot primordia. The control produced 3 parts stems to 1 part leaves; medium with 0.02 mg/l BAP regenerated approximately 2 parts stems and 1 part leaves; that of 0.20 mg/l BAP 1 part stems and 2 parts leaves; and medium with 2.00 mg/l BAP regenerated leaves only. Received: 17 September 1997 / Revision received: 30 October 1997 / Accepted: 18 November 1997