Putrescine and Silver Nitrate Influences Shoot Multiplication, In Vitro Flowering and Endogenous Titers of Polyamines in Cichorium intybus L. cv. Lucknow Local

Abstract The influence of putrescine (Put) and AgNO₃ on shoot multiplication, in vitro flowering and endogenous titers of polyamines in Cichorium intybus L. cv. Lucknow local was investigated. Exogenous administration of Put at a concentration of 40 mM resulted in maximum tissue response in terms of shoot numbers (34.6 ± 2.61) and shoot lengths (7.6 ± 0.57 cm) on MS media supplemented with 2-iP (2.0 mg L⁻¹) and GA₃ (0.5 mg L⁻¹) as observed on the 35^th day. Exogenous application of 40 μM AgNO₃ resulted in maximum shoot number (36.8 ± 2.63) and shoot lengths (7.9 ± 0.76 cm) on day 35 on the same media. Endogenous titers of conjugated spermidine decreased sharply from day 7–21, whereas endogenous conjugated spermine levels peaked on day 28 (1265 ± 94.9 nmoles g⁻¹ FW), after treatment with 40 mM Put. Whereas, AgNO₃ (40 μM) fed samples resulted in higher titers of endogenous conjugated spermine (1405 ± 105.6 nmoles g⁻¹ FW, 3.62 fold over control) on day 14. All other treatments showed decreasing endogenous levels during the whole culture period. Both Put (40 mM) and AgNO₃ (40 μM) resulted in floral initiation and floral development on day 28 and 14 (3.76 ± 0.16, 4.2 ± 0.21 flowers per shoot apices), respectively. To investigate the role of Put (40 mM) and AgNO₃ (40 μM) on morphogenetic response and endogenous conjugated polyamine titers in shoots of chicory, polyamine inhibitors (DFMA and DFMO) were used. The morphogenetic response and the endogenous conjugated pool of polyamines were diminished in DFMA and DFMO treatments, but could be restored by addition of Put (40 mM) and AgNO₃ (40 μM). Under exogenous Put feeding, ethylene production was reduced in shoot cultures of chicory. This study shows for the first time the influence of polyamines on multiple shoot initiation from axillary buds of C. intybus L. cv. Lucknow local and also indicates the promotive effect of Put and AgNO₃ on autoregulation of polyamine biosynthesis, thereby regulating in vitro flowering, the endogenous pool of polyamines and shoot multiplication. Received 12 November 1999; accepted 11 February 2000     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

Limitation of stomatal conductance by hydraulic traits: sensing or preventing xylem cavitation?

We tested the hypothesis that hydraulic conductance per unit leaf surface area of plant shoots (K_SL) determines the maximum diurnal stomatal conductance (g_L) that can be reached by plants growing in the field. A second hypothesis was tested that some xylem cavitation cannot be avoided by transpiring plants and might act as a signal for regulating g_L. Eleven woody species were studied, differing from each other with respect to taxonomy, wood anatomy and leaf habit. Maximum diurnal g_L, transpiration rate (E_L), pre-dawn and minimum diurnal leaf water potential (O_pd and O_min, respectively) were measured in the field. The critical O level at which stem cavitation was triggered (O_cav) was measured on detached branches, using the acoustic method. A high-pressure flow meter was used to measure maximum K_SL of 1-year-old shoots. Both g_L and E_L were positively related to K_SL. The whole-plant hydraulic conductance per unit leaf area (K_WL) of all the species studied, calculated as the ratio of E_L to (O (=O_pd-O_min) was closely related to K_SL. In every case, O_min (ranging between -0.85 and -1.35 MPa in the different species) dropped to the O_cav range or was <O_cav (ranging between -0.71 and -1.23 MPa), thus suggesting that some cavitation-induced embolism could not be avoided. The possibility is discussed that some cavitation-induced reduction in K_SL is the signal for stomatal closure preventing runaway embolism. The lack of correlation of g_L to O_cav is discussed in terms of the inconsistency of O_cav as an indicator of the vulnerability of plants to cavitation. No differences in hydraulic traits were observed between evergreen and deciduous species.     

Putrescine influences growth and production of coumarins in transformed and untransformed root cultures of witloof chicory (Cichorium intybus L. cv. Lucknow local)

The effect of putrescine on growth and production of two coumarins, esculin, and esculetin in the transformed and untransformed roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of putrescine (Put) on growth and production of coumarins, polyamine inhibitors namely α-DL-difluromethylornithine (DFMO) and α-L-difluromethylarginine (DFMA) were used at 1 mM levels. Treatment with 1.5 mM of putrescine (Put) produced 1.96 - fold and 4.0 - fold increase in the growth of transformed and untransformed roots of chicory, respectively. The treatment with polyamine inhibitors showed much lower growth, as well as production compared with both 1.5 mM putrescine treatment and control in both transformed and untransformed chicory roots. The endogenous polyamines, both free and conjugated, were studied over the whole culture period, and it was seen that conjugated titers of all three polyamines viz., putrescine, spermidine and spermine were higher than level of free polyamines, throughout the culture period in both transformed and untransformed roots of chicory. Treatment in which polyamine inhibitors were used showed lower level of endogenous polyamines as compared with the 1.5 mM putrescine treated sample in both the systems. The treatment wherein putrescine was added at 1.5 mM level showed maximum accumulation of endogenous conjugated putrescine (2098.86±157.6 nmoles g⁻¹ FW; 896.8±67.2 nmoles·g⁻¹ FW), on the 14^th day in both transformed and untransformed roots respectively. The production of esculin and esculetin was strictly correlated with growth in every treatment in both systems. Putrescine at 1.5 mM resulted in greater length of primary root in transformed (18.3±1.4 cm) and untransformed (6.86±0.51 cm) as compared with their respective controls (11±0.9 cm; 2.9±0.1cm) and greater number of secondary and tertiary roots. This study suggests that putrescine influences plant root development and differentiation, and it also provides insight into the morphological changes that occur in roots in response to the external supply of polyamines.     

Essential oils enhanced by ultra-high carbon dioxide levels from Lamiaceae species grown in vitro and in vivo

The growth (fresh weight), morphogenesis (leaves, roots and shoots) and essential oil composition of mint (Mentha sp. L.) and thyme (Thymus vulgaris L.) plants were determined after 8 weeks under 350, 1,500, 3,000, 10,000 and 30,000 µmol mol^-1 CO₂. Plants were grown in vitro on basal medium (BM) consisting of Murashige and Skoog salts and 0.8% agar that contained either 0 or 3% sucrose under a 16-h (day)/8-h (night) photoperiod at a light intensity of 180 µmol s^-1 m^-2 or in soil in a greenhouse under conditions of natural sunlight. Ultra-high CO₂ levels (i.e. ́,000 µmol mol^-1 CO₂) substantially increased fresh weights, leaves, shoots and roots for all plants compared to plants grown under ambient air (350 µmol mol^-1 CO₂) both in vivo and in vitro. For both species, 10,000 µmol mol^-1 CO₂ was the optimum concentration to obtain the largest growth and morphogenesis responses under in vitro conditions, while the 3,000- to 10,000-µmol mol^-1 CO₂ range provided the largest yields for soil-grown plants. Essential oil composition (i.e. monoterpenes, piperitonone oxide and limonene from mint and aromatic phenol and thymol from thyme) from the shoot portion of plants grown at all CO₂ levels was analyzed in CH₂Cl₂ extracts via gas chromatography. Higher levels of secondary compounds occurred in vitro when cultures were grown under ultra-high CO₂ levels than in ambient air. The concentration of thymol, a major secondary compound in thyme plants grown on BM containing sucrose, was 317-fold higher at 10,000 µmol mol^-1 CO₂ than in plants grown under ambient air conditions with the same BM. The levels of secondary compound in in-vitro-grown plantlets exposed to ultra-high CO₂ concentrations exceeded those occurring in plants grown in the greenhouse under the same CO₂ levels. Substantially higher levels of secondary compound occurred in plants under ultra-high CO₂ levels on BM containing sucrose than on BM lacking sucrose or in soil. Thymol levels in thyme plants grown on BM containing sucrose were 3.9-fold higher at 10,000 µmol mol^-1 CO₂ than in shoots grown on BM without sucrose under the same CO₂ levels. High positive correlations occurred between thymol concentrations and CO₂ levels, fresh weights, shoots, roots and leaves when thyme shoots were grown on BM with sucrose. High positive correlations for thyme shoots grown on BM without sucrose only occurred between thymol concentrations and CO₂ levels, fresh weights, shoots and leaves. No positive correlations between thymol concentrations and CO₂ levels or any growth or morphogenesis responses occurred for thyme shoots when grown in soil.     

Clonal propagation of Lilium nepalense D.Don, a threatened medicinal plant of Nepal

A protocol for the micropropagation of Lilium nepalense D.Don, a highly prized medicinal plant of Nepal, has been developed. Axillary shoots were regenerated from twin-scale explants prepared from mature bulbs. Multiplication was carried out on MS medium supplemented with 20 µM zeatin using longitudinally split shoot halves. On average, more than seven shoots were obtained from one explant in a 4-week culture period. After rooting the cloned plantlets were successfully hardened to ex vitro conditions. Preliminary trials in Nepal showed that the method can be successfully applied for the production of plants suitable for field cultivation. This may be an alternative to the over-exploitation of the natural resources of the species.     

Influence of modified oxygen and carbon dioxide atmospheres on mint and thyme plant growth, morphogenesis and secondary metabolism in vitro

. Growth (fresh weight) and morphogenesis (production of leaves, roots and shoots) of mint (Mentha sp. L.) and thyme (Thymus vulgaris L.) shoots were determined under atmospheres of 5%, 10%, 21%, 32%, or 43% O₂ with either 350 or 10,000 µmol mol^-1 CO₂. Plants were grown in vitro on Murashige and Skoog salts, 3% sucrose and 0.8% agar under a 16/8-h (day/night) photoperiod with a light intensity of 180 µmol s^-1 m^-2. Growth and morphogenesis responses varied considerably for the two plant species tested depending on the level of O₂ administered. Growth was considerably enhanced for both species under all O₂ levels tested when 10,000 µmol mol^-1 CO₂ was added as compared to growth responses obtained at the same O₂ levels tested with 350 µmol mol^-1 CO₂. Mint shoots exhibited high growth and morphogenesis responses for all O₂ levels tested with 10,000 µmol mol^-1 CO₂. In contrast, thyme shoots exhibited enhanced growth and morphogenesis when cultured in ₁% O₂ with 10,000 µmol mol^-1 CO₂ included compared to shoots cultured under lower O₂ levels. Essential oil compositions (i.e. monoterpene, piperitenone oxide from mint and aromatic phenol, thymol from thyme) were analyzed from CH₂Cl₂ extracts via gas chromatography from the shoot portion of plants grown at all O₂ levels. The highest levels of thymol were produced from thyme shoots cultured under 10% and 21% O₂ with 10,000 µmol mol^-1 CO_2,and levels were considerably lower in shoots grown under either lower or higher O₂ levels. Higher levels of piperitenone oxide were obtained from mint cultures grown under ₁% O₂ with 10,000 µmol mol^-1 CO₂ compared to that obtained with lower O₂ levels.     

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20 min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2 M sucrose solution for 20 min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10 days of plating and developed shoots within 3 weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.     

Regeneration of Lonicera tatarica plants via adventitious organogenesis from cultured stem explants

We describe an efficient process for the regeneration of Lonicera tatarica plants from cultured stem sections. Induction of multiple shoots was achieved directly from cultured stem cuttings. The highest regeneration rate was achieved on Gamborg's B5 medium supplemented with 4% sucrose and 0.8% Difco bacto-agar in the absence of hormones. Differentiated shoots were elongated for 5-7 days on induction medium supplemented with 0.5 mg/l GA₃. Shoot induction and elongation experiments were carried out using original stem explants from either 2-, 6-, or 18-month-old donor plants. The age of the donor plant had no noticeable effect on either process. However, rooting of elongated shoots occurred only with shoots derived from 2-month-old donor plants. Rooting efficiency and proliferation were highest on half-strength WPM medium supplemented with 2 µM indole-3-butyric acid and 0.6% Keylis agar. The plants regenerated from stem explants were morphologically normal, and levels of loganin and secologanin were comparable to those detected in plants grown from seed and maintained through vegetative propagation.     

Methyl laurate and 6-benzyladenine promote the germination of somatic embryos of a hybrid rose

Globular stage somatic embryos were induced in callus cultures of Rosa Heritage 2 Alister Stella Gray on medium containing 13.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and developed to the cotyledonary stage on medium containing 9 µM 2,4-D. Cotyledonary-stage embryos were transferred to germination media with or without 1.5 µM 6-benzyladenine (BA) and with or without 44 µM methyl laurate (Mela). BA and Mela both promoted the development of shoots and roots and increased the frequency of bipolar germinations. An average of 56.5% (SEdž.1%) embryos on medium containing both BA and Mela underwent bipolar germinations compared with less than 20% in treatments where either or both were excluded. The effectiveness of BA and Mela was reduced if Mela was included in the development medium or if the concentration of salts and vitamins in the germination media was sub-optimal. There was evidence that growth at one pole of the somatic embryo promoted development at the other.     

Germination of synthetic seeds of pineapple (Ananas comosus L. Merr.)

. Tufts of multiple shoots were produced from dormant, axillary buds of pineapple in vitro. Tiny shoots (2-5 mm) isolated from the tuft of multiple shoots were encapsulated in 3% sodium alginate prepared using hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose. The encapsulated shoots represented synthetic seeds that germinated and formed roots in vitro after subculture onto one of the following media solidified with 0.8% agar: (1) hormone-free Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol and 0.06 M sucrose (Pin1), (2) Murashige and Skoog's basal medium, Murashige and Skoog's vitamins, 0.56 mM myo-inositol, 0.06 M sucrose, 9.67 µM 1-naphthalene acetic acid, 9.84 µM indole-3-butyric acid and 9.29 µM kinetin (Pin2), and (3) White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 0.54 µM 1-naphthalene acetic acid and 1.97 µM indole-3-butyric acid (Pin3). Pretreatment of shoots in either liquid Pin3 or Pin4 medium (White's basal medium, White's vitamins, 0.56 mM myo-inositol, 0.03 M sucrose, 10.8 µM 1-naphthalene acetic acid and 39.4 µM indole-3-butryic acid) was required for development into plantlets with roots after culture on either Pin1, Pin2 or Pin3 media. One hundred percent germination of synthetic seeds to plantlets occurred after pretreatment of shoots in liquid Pin4 medium for 12 h followed by culture of synthetic seeds on Pin2 medium. Synthetic seeds stored at 4°C remained viable without sprouting for up to 45 days. Plantlets produced in vitro from synthetic seeds were successfully established in soil. The protocol provides an easy and novel propagation system for pineapple, an otherwise vegetatively propagated fruit crop.     

Cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase: a novel enzyme-based selectable marker for plant transformation

The mutant glutamate-1-semialdehyde aminotransferase (GSA-AT) enzyme encoded by the hemL gene of the gabaculine-resistant cyanobacterium Synechococcus PCC6301 strain GR6 was expressed in tobacco following Agrobacterium-mediated transformation of leaf discs. When targeted to plastids, the GR6 hemL gene product conveyed gabaculine resistance to transgenic plants. Selection using 50 and 100 µM gabaculine was shown to produce two distinct explant phenotypes: 'greens' and 'whites'. T₁ progeny displayed Mendelian segregation ratios, and PCR analysis demonstrated the 'green' phenotype corresponded with the presence of the GR6 hemL gene. Furthermore, 'whites' could be rescued after 9 days growth on solid media containing between 5 µM and 25 µM gabaculine, allowing the potential use of this system for the isolation of gabaculine-sensitive transformants in mutagenesis screening. The use of GR6 hemL as a selectable marker gene provides a novel enzyme-based method for the selection of transgenic regenerants without the need for antibiotic-resistance markers.     

Physiological Controls on the Production of Cleistogamous and Chasmogamous Flowers in Lamium amplexicaule L

Plants of Lamium amplexicaule, grown under short-day field conditionsin Northern California produce predominantly closed flowers.Under long-day field conditions, plants may produce up to 50per cent open flowers, the same total number of flowers beingproduced under each daylength regime. A 20 ml drop of GA₃ or GA₇ (100µM) was applied to themain shoot apex of plants growing under short-day conditions,and all subsequently produced flowers opened. CCC, an inhibitorof gibberellin synthesis, was applied (0.06 per cent) to thesoil of seedlings grown under long-day conditions. The CCC-treatedplants were dwarfed and bore only closed flowers. With GA₇ appliedexogenously to the CCC-treated shoots, inhibition was released,resulting in elongated internodes and open flower production.The timing of flower production and internodal extension inLamium amplexicaule are positively correlated. When floral primordiawere removed from main shoots, the average internode lengthsdecreased. The number of nodes produced in treated plants wasincreased as a result of flower removal. Exogenous GA₇ (10 µM)applied to the nodes from which flowers were removed resultedin internodal extension but had no effect on node number. Twoprocesses that may contribute to the control of the productionof open and closed flowers in Lamium amplexicaule are: (1) anincreasing anther sac size from lower to upper node flowersthat may exert control locally, via GN production, on corollaexpansion, and (2) a photoperiodic control. Lamium amplexicaule L, henbit, cleistogamy, chasmogamy, gibberellins, gibberellic acid (GA₃), (2 chloroethyl) trimethylammonium chloride (cycocel)     

Water relations of coastal and estuarine Rhizophora mangle: xylem pressure potential and dynamics of embolism formation and repair

Physiological traits related to water transport were studied in Rhizophora mangle (red mangrove) growing in coastal and estuarine sites in Hawaii. The magnitude of xylem pressure potential (P_x), the vulnerability of xylem to cavitation, the frequency of embolized vessels in situ, and the capacity of R. mangle to repair embolized vessels were evaluated with conventional and recently developed techniques. The osmotic potential of the interstitial soil water (?_sw) surrounding the roots of R. mangle was c. -2.6LJ.52᎒^-3 and -0.4Lj.13᎒^-3 MPa in the coastal and estuarine sites, respectively. Midday covered (non-transpiring) leaf water potentials (O_L) determined with a pressure chamber were 0.6-0.8 MPa more positive than those of exposed, freely-transpiring leaves, and osmotic potential of the xylem sap (?_x) ranged from -0.1 to -0.3 MPa. Consequently, estimated midday values of P_x (calculated by subtracting ?_x from covered O_L) were about 1 MPa more positive than O_L determined on freely transpiring leaves. The differences in O_L between covered and transpiring leaves were linearly related to the transpiration rates. The slope of this relationship was steeper for the coastal site, suggesting that the hydraulic resistance was larger in leaves of coastal R. mangle plants. This was confirmed by both hydraulic conductivity measurements on stem segments and high-pressure flowmeter studies made on excised leafy twigs. Based on two independent criteria, loss of hydraulic conductivity and proportions of gas- and liquid-filled vessels in cryo-scanning electron microscope (cryo-SEM) images, the xylem of R. mangle plants growing at the estuarine site was found to be more vulnerable to cavitation than that of plants growing at the coastal site. However, the cryo-SEM analyses suggested that cavitation occurred more readily in intact plants than in excised branches that were air-dried in the laboratory. Cryo-SEM analyses also revealed that, in both sites, the proportion of gas-filled vessels was 20-30% greater at midday than at dawn or during the late afternoon. Refilling of cavitated vessels thus occurred during the late afternoon when considerable tension was present in neighboring vessels. These results and results from pressure-volume relationships suggest that R. mangle adjusts hydraulic properties of the water-transport system, as well as the leaf osmotic potential, in concert with the environmental growing conditions.     

In vitro induction of tetraploid <Emphasis Type="Italic">Ziziphus jujuba</Emphasis> Mill. var. <Emphasis Type="Italic">spinosa</Emphasis> plants from leaf explants

The genetic manipulation of Capsicum has been unsuccessful, and a large bottleneck to transferring the desired genes is due to the difficulty in regenerating whole plants through tissue culture because of its highly recalcitrant and high genotype specificity. This study aimed to investigate and establish rapid shoot regeneration from the proximal ends of the leaves of Capsicum frutescens KT-OC and BOX-RUB varieties. A maximum of 8–10 shoot buds were obtained from the margins of the proximal portion of a cotyledonary leaf explant of C. frutescens variety KT-OC on medium I containing 44.44 µM 6-benzylaminopurine (BA), 5.71 µM indole-3-acetic acid (IAA), 10 µM silver nitrate (AgNO₃) and 1.98 mg L^?1 2-(N-morpholine) ethane sulphonic acid within 4 weeks of incubation, of which 60% of explants responded in terms of shoot buds. Petiole explants (40%) cultured on the same medium produced 2–4 shoots per explant from the distal portion. The cut portions of the cotyledonary leaf proximal portions responded well to shoot bud formation in the presence of 22.20 µM BA and 14.68 µM phenyl acetic acid (PAA), wherein 100% of explants responded in terms of shoot bud formation, with an average of 10?±?1.7 and 8?±?1.9 shoot buds per explant in KT-OC and BOX-RUB varieties, respectively. The differentiated shoots grew well and proliferated in the presence of 14.68 µM PAA?+?22.20 µM BA and 10 µM AgNO₃. Shoot elongation was obtained in presence of 1.44 µM gibberellic acid (GA₃) and 10 µM AgNO₃. These shoots were rooted on plant growth regulator-free half-strength MS medium and upon hardening; field survival rate was 70%. This reproducible regeneration method for C. frutescens, especially the Indian high pungent variety, from proximal portion of cotyledonary leaf and petiole explants, can be used for biotechnological improvement.     

Growth and Partitioning in Pascopyrum smithii (C3) and Bouteloua gracilis(C4) as Influenced by Carbon Dioxide and Temperature

This study investigated how CO₂and temperature affect dry weight(d.wt) accumulation, total nonstructural carbohydrate (TNC)concentration, and partitioning of C and N among organs of twoimportant grasses of the shortgrass steppe,Pascopyrum smithiiRydb. (C₃) andBouteloua gracilis(H.B.K.) Lag. ex Steud. (C₄).Treatment combinations comprised two temperatures (20 and 35°C)at two concentrations of CO₂(380 and 750 µmol mol^-1),and two additional temperatures of 25 and 30°C at 750 µmolmol^-1CO₂. Plants were maintained under favourable nutrient andsoil moisture and harvested following 21, 35, and 49d of treatment.CO₂-induced growth enhancements were greatest at temperaturesconsidered favourable for growth of these grasses. Comparedto growth at 380 µmol mol^-1CO₂, final d.wt of CO₂-enrichedP.smithiiincreased 84% at 20°C, but only 4% at 35°C. Finald.wt ofB. graciliswas unaffected by CO₂at 20°C, but wasenhanced by 28% at 35°C. Root:shoot ratios remained relativelyconstant across CO₂levels, but increased inP. smithiiwith reductionin temperature. These partitioning results were adequately explainedby the theory of balanced root and shoot activity. Favourablegrowth temperatures led to CO₂-induced accumulations of TNCin leaves of both species, and in stems ofP. smithii, whichgenerally reflected responses of above-ground d.wt partitioningto CO₂. However, CO₂-induced decreases in plant tissue N concentrationswere more evident forP. smithii. Roots of CO₂-enrichedP. smithiihadgreater total N content at 20°C, an allocation of N below-groundthat may be an especially important adaptation for C₃plants.Tissue N contents ofB. graciliswere unaffected by CO₂. Resultssuggest CO₂enrichment may lead to reduced N requirements forgrowth in C₃plants and lower shoot N concentration, especiallyat favourable growth temperatures. Acclimation to CO₂; blue grama; Bouteloua gracilis ; carbohydrate; climate change; global change; grass; growth; growth temperature optima; nitrogen; N uptake; Pascopyrum smithii; western wheatgrass     

Growth of Lucerne (Medicago sativa L.) Exposed to Sulphur Dioxide

The effect of exposure to sulphur dioxide (0 or 96 µgm^–3 SO₂) on the growth and sulphur content of lucerne(Medicago sativa L.) was examined in a period of 135 d duringwhich the plants were harvested four times. The lucerne wasgrown in pots of soil with and without the addition of sulphateand of nitrate. Evidence of sulphur deficiency, including areduction in the weight, the number and the sulphur contentof shoots, was found in plants grown without added sulphate.Deficiency was alleviated through exposure of plants to SO₂.Apart from reducing shoot weight at one harvest and generallyincreasimg the concentration of sulphur in the shoots, exposureto SO₂ had no significant effect on plants grown with addedsulphate. The yield of shoots was greater, and was reduced toa lesser extent with sulphur deficiency, in plants grown withadded nitrate than in those dependent on rhizobia. Whilst thetranspiration coefficient increased greatly in sulphur-deficientplants, it was reduced where otherwise similarly treated plantswere exposed to SO₂; this treatment did not alter the coefficientin plants with an adequate supply of sulphur from the soil.     

Influence of Ni Supply on Growth and Nitrogen Metabolism ofBrassica napusL. Grown with NH4NO3or Urea as N Source

The significance of nickel (Ni), which is essential for ureaseactivity, for growth and nitrogen (N) metabolism ofBrassicanapusgrown in nutrient solution with either NH₄NO₃or urea assole N source was investigated. Although Ni contents were below25 µg kg^-1d. wt, growth of plants relying on NH₄NO₃wasnot affected by the Ni status. However, supplementing the growthmedium with 0.04 µMNi enhanced dry matter production ofurea-grown plants significantly. Urease activity was significantlyreduced in leaves and roots of plants grown without supplementaryNi irrespective of N source. Plants grown with urea withoutadditional Ni accumulated large amounts of urea and had loweramino acid contents indicating impaired usage of the N supplied,while those grown with NH₄NO₃under Ni-deprived conditions accumulatedendogenous urea in their older leaves. It is suggested thatNi may not be strictly essential for plants receiving mineralN, or that the critical level is well below 25 µg kg^-1d.wt. These results confirm that Ni is required for urease activityand thus for growth of plants on urea-based media, as well asfor recycling endogenous urea.Copyright 1999 Annals of BotanyCompany. Brassica napusvar.annua, amino acids, N nutrition, nickel, spring rape, urea, urease activity.     

Effect of silver nitrate on sugarcane cell suspension growth, protoplast isolation, ethylene production and shoot regeneration from cell suspension cultures

Silver nitrate (AgNO₃), an inhibitor of the physiological actionof ethylene, reduced cell growth, promoted ethylene production,increased the yield of protoplasts and reduced shoot regenerationfrom sugarcane heterogeneous cell suspension cultures. The increasein the rate of protoplast isolation from cultures treated withAgNO₃ (0 to 59 µM) correlate with an increase in endogenousethylene production by the cells. The addition to the culturemedium of chemicals that either inhibited (aminoethoxyvinylglycine,AVG) or promoted (aminocyclopropane-1-carboxylic acid, ACC)ethylene biosynthesis did not alter the number of protoplastsisolated from these cultures. However, protoplasts were isolatedwith AVG in combination with AgNO₃ even though ethylene productionwas inhibited. These results suggested that AgNO₃ may be havinganother more direct effect on protoplast release. One such sitemay be the cell wall or on cell metabolism conditioning cellsto release protoplasts after enzyme treatment. Key words: Sugarcane, cell suspension, protoplast, silver nitrate, ethylene