Changes in macromolecular movement accompany organogenesis in thin cell layers of <Emphasis Type="Italic">Torenia fournieri</Emphasis>

A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)₃: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.     

Influence of putrescine, silver nitrate and polyamine inhibitors on the morphogenetic response in untransformed and transformed tissues of Cichorium intybus and their regenerants

The addition of 40 mM putrescine (Put) to Murashige and Skoog's (MS) medium resulted in increased shoot multiplication and shoot growth in untransformed plants relative to transformed plants of Cichorium intybus L. Put at a concentration of 40 mM also resulted in flowering in both systems on the 28th day, with elevated titers of endogenous conjugated Put and spermine (Spm) in both untransformed and transformed plants. The addition of 40 µM AgNO₃ to untransformed axillary buds of C. intybus L. cultured on MS media resulted in increased shoot multiplication (36.9DŽ.63 shoots per culture) and increased shoot growth (7.82ǂ.76 cm) as compared to transformed ones (11.6ǂ.89 shoots per culture; 3.20ǂ.24 cm). Moreover, cultures treated with 40 µM AgNO₃ showed in vitro flowering on the 28th day in both systems, with the endogenous levels of conjugated spermine being higher in untransformed plants than in transformed ones. The morphogenetic response and the endogenous conjugated pool of polyamines were lower following !-DL-difluromethylarginine and !-DL-difluromethylornithine treatments; the addition of put (40 mM) and AgNO₃ (40 µM) restored these to normal levels. Under exogenous put feeding, ethylene production was lower in both the untransformed and transformed cultures. We believe that an interplay between polyamine and ethylene biosynthesis is involved in regulating the morphogenetic response in both transformed and untransformed shoots of C. intybus. The response to AgNO₃ and Put treatment was not altered by the transformation process.     

Symplast domains in extrastelar tissues of Egeria densa Planch.

A set of hydrophilic fluorescent dyes of known molecular weight has been used to determine the molecular exclusion limit and the extent of apical, epidermal and cortical symplasts in the root, stem and leaf of Egeria densa. These dyes are unable to pass the plasmalemma, so that any cell-to-cell movement of injected dye must occur via the symplast. The shoot-apex symplast has a high molecular exclusion limit, excluding dyes with a molecular weight of 749 dalton (fluorescein hexaglycine) and greater but allowing dyes of up to 665 dalton (fluorescein diglutamic acid) to pass. The leaf epidermal symplast is similar to that in the apex: fluorescein pentaglycine (674 dalton) moves to a limited extent, but fluorescein hexaglycine is immobile. Stem and root epidermal cells have a lower molecular exclusion limit, only the dye 6-carboxyfluorescein (376 dalton) is able to move from cell-to-cell. Cortical and epidermal tissues in both the stem and the root have similar symplast permeabilities. However, a barrier to dye (6-carboxyfluorescein) movement is found between the epidermis and the cortex in both organs. Barriers are also found at the nodes between expanded internodes. The stem barriers are not found in the unexpanded nodes near the shoot tip; apparently they are formed early during internode expansion. In the root tip, a barrier to the movement of dye is found between the root cap and the remainder of the root. Plasmodesmata are found linking all cell types studied, even cells where barriers to dye movement occur. Thus, the plant, far from being one uniform symplast, consists of a large number of symplast domains, which may or may not differ in molecular exclusion limit.Abbreviations F fluorescein isothiocyanate isomer I - Glu l-glutamic acid - (Glu)₂ l-glutamylglutamic acid - (Gly)₅ l-pentaglycine - (Gly)₆ l-hexaglycine     

Histological analysis of somatic embryogenesis and adventitious shoot formation from root explants of <Emphasis Type="Italic">Centaurium erythreae</Emphasis> Gillib

Direct somatic embryogenesis and adventitious shoot formation were successfully achieved from root explants of Centaurium erythrea Gillib. cultured on Murashige and Skoog medium with half-strength macronutrients, full-strength micronutrients and vitamins, 3 % sucrose, 0.7 % agar, 100 mg dm⁻³ myo-inositol and without growth regulators. Histological studies revealed that somatic embryos were formed directly from epidermal cells and adventitious buds were developed from meristematic cells in root cortex tissues. Somatic embryos as well as adventitious shoots developed into whole plantlets.     

Activity of antioxidant enzymes and secondary metabolites during in vitro regeneration of Sterculia urens

The changes in the activities of antioxidant enzymes and amounts of proteins, phenols, and flavonoids in regenerating and non-regenerating calli during organogenesis of Sterculia urens were monitored. Maximum growth of calli and the most efficient regeneration of shoots occurred on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^?3 benzylaminopurine (BAP) + 2 or 4 mg dm^?3 naphtalene acetic acid (NAA). Peroxidase (POD), catalase, and superoxide dismutase activities increased in the regenerating calli but decreased in the non-regenerating calli. Six POD isoenzymes were detected. Protein content decreased in the non-regenerating calli and increased significantly during regeneration of shoots from callus. Total phenols and flavonoids increased in the non regenerating calli. SDS-PAGE analysis revealed a role of many proteins in organogenesis.     

Quantitative analyses of shade-shoot architecture of conifers native to New Zealand

Shoot architecture was quantified by measuring the "maximum silhouette area ratio" (R_max). R_max was calculated from the maximum silhouette area (or projected area) of the intact shoot, divided by the silhouette area of the leaves or phylloclades (leaf-like flattened stems) when they are removed from the shoot and laid out flat. Like conifers of the Northern Hemisphere (NH) with non-appressed foliage, the R_max of shade-adapted shoots ranged from 0.5 to 1.0 in New Zealand (NZ) conifers with non-appressed foliage. Defining a "leaf" to mean either a true leaf or a phylloclade, the following was found: leaf area/leaf dry weight, leaf area/shoot dry weight, and leaf dry weight/shoot dry weight, were all similar in the shade-shoots of NZ and NH conifers. None of these variables were significantly correlated with R_max in the NZ conifers, unless species with leaves averaging less than 4 mm² in size were excluded from the analyses. Foliage dry weight/shoot projected area was strongly correlated with R_max. NZ conifers had both smaller and larger mean leaf sizes in comparison to NH conifers. The mean projected area per shade-adapted leaf of NZ conifers varied from 2.7 to 436 mm². In NH conifers, the mean projected area per shade leaf varied from 12 to 83 mm². Except for the strikingly larger range in leaf size in NZ conifers, the data support a hypothesis of strong convergent evolution of shade-shoot architecture in NZ and NH conifers. The results are discussed in relation to photosynthesis, stand production, and the ecological distribution of conifers.     

Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis

Efficient protocols of axillary bud multiplication and indirect organogenesis were established for Holostemma ada-kodien Schult. (Asclepiadaceae). Murashige and Skoog (MS) medium supplemented with 2.0 mg l^-1 N⁶-benzylaminopurine (BAP) and 0.5 mg l^-1 indole-3-butyric acid (IBA) induced an average of eight shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. The explant source of callus and the growth regulator inducing the callus exhibited significant influence on organogenesis. Callus developed from the basal cut end of the node explants differentiated more than 15 shoots on MS medium fortified with 1.5 mg l^-1BAP. Callus from internode explants developed fewer shoots than callus from the basal cut ends of node explants. Leaf-derived callus did not undergo organogenesis. The abscission of leaves and shoot tips of the developed shoots was prevented by the addition of AgNO₃ or CoCl₂, but with a concomitant significant reduction in the number of shoots. Half-strength solid MS or liquid medium with 0.05 mg l^-1 IBA exhibited the best in vitro rooting. Ninety percent of the rooted shoots survived in the field.     

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.     

TDZ, auxin and genotype effects on leaf organogenesis in Fragaria

The different types of organogenic (roots and adventitious shoots) and callus formation responses of leaves from 30-day-old proliferating shoots of different Fragaria spp. genotypes were studied in response to MS medium supplemented with 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron; TDZ) alone and in combination with 0.98 μM indole-3-butyric acid (IBA), 0.84 μM3-benzo[b]selenienyl acetic acid (BSAA) or 0.90 μM2,4-dichlorophenoxy acetic acid (2,4-D). The study included: nine octoploid Fragaria x ananassa cultivars and breeding selections; two octoploid breeding selections from F. virginiana glauca inter-species crosses; two diploid F. vesca cultivars; and one diploid clone of F. nubicola Lindl. TDZ plus IBA promoted the highest shoot regeneration efficiencies from leaves of nearly all of the genotypes, while the TDZ/BSAA and TDZ/2,4-D combinations promoted high regeneration efficiencies for only some of the genotypes (Alpina W.O., Sveva, AN 91.371.53, Onda, Paros and FO93.143.5). For the more efficient regenerating genotypes, IBA induced the highest frequency of regenerating leaves, while BSAA induced the highest number of regenerated shoots from leaves and more callus production for most of the genotypes.     

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.     

Relations between efficiency of water transport and duration of leaf growth in some deciduous and evergreen trees

Shoot and leaf growth rate as well as shoot hydraulic conductance per unit leaf area (K_SL) were measured on three evergreen (Viburnum tinus L., Prunus laurocerasus L., Laurus nobilis L.) and three deciduous (Corylus avellana L., Juglans regia L., Castanea sativa L.) trees growing under the same environmental conditions. The times required to complete shoot growth (27 days for P. laurocerasus to 51 days for V. tinus) and leaf expansion (24 days for C. sativa to 42 days for C. avellana) were very different among the studied species. These species also differed in K_SL that ranged between 1.5 and 3.5 e-4 kg s^-1 m^-2 MPa^-1 in C. avellana and C. sativa, respectively, with intermediate values recorded in the other species. A strong, negative and statistically significant correlation was found to exist between K_SL and the time required for complete leaf expansion. This suggests that duration of leaf growth is shortened by the high hydraulic efficiency of the shoot. In contrast, no statistically significant relationship was found to exist between K_SL and shoot growth rate. Whether a high leaf growth rate can be interpreted as advantageous to plants or it is only an epiphenomenon of the high efficiency in the vertical water transport is discussed.     

Histology and chimeral segregation reveal cell-specific differences in the competence for shoot regeneration and Agrobacterium-mediated transformation in Kohleria internode explants

Summary Internode explants of Kohleria sp. (Gesneriaceae) are capable of regenerating large numbers of adventitious shoots. Regeneration of green shoots from explants of an albino periclinal chimera with genetically green L1, as well as microsurgical removal of the epidermis revealed that shoots originate only from the epidermis. Histological studies further showed that shoots arise from a particular epidermal cell type, viz the basal cell of young glandular trichomes. On the other hand, cells competent for Agrobacterium-mediated transformation are mainly located in vascular tissues, as could be shown by histochemical localization of ß-glucuronidase (GUS) expression in explants that had been inoculated with A. tumefaciens strains carrying binary plasmids with GUS and kanamycin resistance (NPTII) genes. Only 3% of GUS expression events took place in the epidermis. Consequently, shoot regeneration in the presence of kanamycin was very poor. Moreover, most of those shoots proved GUS-negative and did not survive subcultivation on kanamycin-containing medium. Six regenerants, however, were most probably transgenic, as suggested by the ability to produce adventitious shoots in the presence of kanamycin and by polymerase chain reaction (PCR) analysis. To our knowledge, this is the first positive result towards genetic transformation in a taxon of the Gesneriaceae.Abbreviations BA N⁶-benzyladenine - ct cefotaxime - GUS ß-glucuronidase - IAA indole-3-acetic acid - km kanamycin - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction     

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.     

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.     

Genotypic variation in the response of two perennial grass species to elevated carbon dioxide

Shoot and reproductive biomass of genotypes of Bromus erectus and Dactylis glomerata grown in competition at ambient and elevated CO₂ were examined for 2 consecutive years in order to test whether genetic variation in those traits exists and whether it is maintained over time. At the species level, a positive CO₂ response of shoot biomass of both species was only found in the first year of treatment. At the genotype level, no significant CO₂2genotype interaction was found at any single harvest either for vegetative or reproductive biomass of either species. Analysis over time, however, indicated that there is a potential for evolutionary adaptation only for D. glomerata: (1) repeated measures ANOVA detected a marginally significant CO₂2genotype2time interaction for shoot biomass, because the range of the genotypes CO₂ response increased over time; (2) genotypes that displayed the highest response during the first year under elevated CO₂ also showed the highest response the second year. Null (B. erectus) or weak (D. glomerata) selective potentials of elevated CO₂ were detected in this experiment, but short time series could underestimate this potential with perennial species.     

Effects of Waterlogging on the Gibberellin Content and Growth of Tomato Plants

Flooding of tomato roots results in decreased stem growth. Wehave shown that flooding will reduce levels of gibberellins(GA) in the roots, shoots, and bleeding sap of tomato plants.The adventitious roots that appear on the third day of waterloggingmay be responsible for the production of GA that accumulatein the shoot after 3 to 4 days of flooding. The endogenous GAof tomato will stimulate stem growth of tomato plants. Initially,application of gibberellic acid (GA₃) will stimulate the growthof flooded plants to a greater extent than that of nonwaterloggedplants. It is suggested that one of the first effects of floodingis to reduce GA levels and so inhibit stem elongation. At alater stage of waterlogging GA₃ is less effective and otherfactors appear to inhibit shoot growth.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L.

We have developed a highly efficient three-stage protocol for plant regeneration in sunflower (Helianthus annuus L.) from embryonal cotyledons. This protocol uses phenylacetic acid (PAA) for both shoot-bud induction and the elongation of smaller buds. The medium used for inducing bud formation from the cotyledons was modified MS medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l PAA. Buds were elongated on MS medium supplemented either with only 0.2 mg/l gibberellic acid (GA₃) or with 0.2 mg/l GA₃ + 0.1 mg/l PAA + 0.3 mg/l BAP. The elongated shoots were then transferred onto rooting medium containing 1 mg/l PAA. The complete plantlets with well-developed roots were transferred to field conditions where they survived and set normal seeds. The induction of shoot buds from embryonal cotyledons was also observed on modified MS medium supplemented with 0.5-5 mg/l BAP in combination with 0.5-5 mg/l !-naphthaleneacetic acid (NAA). In this case, the formation of callus took place along with shoot-bud formation, which hindered further development of the latter. The presence of PAA with BAP in the primary bud induction medium promoted normal development and elongation of shoot buds.     

Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures

Experiments were performed to determine the effects ofcarbon source and concentration on shootmultiplication in shoot cultures of Fagussylvatica (one clone) and F. orientalis (twoclones) and on the induction of adventitious shootbuds from leaf and internode explants of F.orientalis. In general, glucose was the best carbonsource for both axillary branching and adventitiousshoot regeneration. Shoot-tip explants grown on 3–4%glucose medium produced more shoots than those onsucrose or fructose. For maximum shoot length, glucosemedium was best for two of the three clones, and 4%sucrose for the other. The number of shoots was theparameter most influenced by glucose concentration inthe adventitious shoot regeneration experiments, thenumber increasing with sugar concentration. The lowesthyperhydricity rate occurred in the presence ofsucrose in both species. Shoot growth and quality wasnegatively affected by fructose supplied media. Theuse of filter-sterilized rather than autoclavedfructose neither stimulated shoot growth nor reducedthe incidence of hyperhydricity in all three clones.The response of shoot cultures to differentcarbohydrate treatments appears to some extent to begenotype dependent.     

Patterns of morphogenesis from cotyledon explants of pigeonpea

Summary Cotyledons excised from seedlings of Cajanus cajan (pigeonpea) were grown on media containing cytokinins (6-benzyladenine, zeatin, and zeatin riboside) and an allied compound, thidiazuron. With the exception of zeatin riboside, initial response in terms of induction of organized structures was very high. However, subsequent regeneration of shoots from cotyledon explants was very poor. Anatomical studies on the regenerating explants were undertaken to study the pattern of morphogenesis. Cytokinins and thidiazuron induced divisions in the epidermal and sub-epidermal cell layers leading to the formation of primary protrusions on the surface. This was followed by the development of foci of high meristematic activity either on the surface or within the primary protrusions. These foci differentiated into embryo-like structures or shoot meristem-like structures. Mostly aberrant shoots, with poorly developed apical meristems, regenerated from these structures.     

Age-Dependent Changes in Levels of Ascorbic Acid and Chlorogenic Acid, and Activities of Peroxidase and Superoxide Dismutase in the Apoplast of Tobacco leaves: Mechanism of the Oxidation of Chlorogenic Acid in the Apoplast

In order to understand browning in tobacco plants during aging,age-dependent changes in the levels of ascorbic acid (AA) andchlorogenic acid (CGA) and its isomers were investigated inthe apoplast and the symplast of the leaves. Also activitiesof peroxidase (POX) and superoxide dismutase (SOD) were determined.AA decreased during aging until it was no longer detectablein the apoplast, while symplastic AA remained although the leveldecreased on aging. In contrast, levels of CGA and its isomersand activity of POX in the apoplast increased on aging, whilethose in the symplast remained nearly constant in mature andold leaves. The activity of SOD in the apoplast increased duringaging, while that in the symplast decreased. Oxidation of CGAby the apoplastic solution was observed in the absence of externallyadded H₂O₂ and the oxidation was inhibited by SOD and catalase.Brown components, which contained caffeic acid moieties, accumulatedin the apoplast on aging and the components produced O–₂and H₂O₂ by autooxidation. From these results, we conclude (i)that brown components are formed in the apoplast by the CGA/POXsystem, (ii) that the H₂O₂ required for the reaction can beprovided by the CGA/POX system itself and by autooxidation ofthe brown components, and (iii) that apoplastic SOD functionsto generate H₂O₂ from apoplastically formed O–₂. (Received February 8, 1999; Accepted May 7, 1999)