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BACKGROUND: Several studies have shown that cherries, especially sour cultivars, contain substantial amounts of phenolic compounds. OBJECTIVE: This study aims to (i) analyze the total phenolic compound (TPC) content and the antioxidant capacity (AC) of a large range of cultivars using the same methodology in one laboratory, and (ii) determine the possible relationship between agronomic characteristics and AC. METHODS: A total of 245 samples including sweet, sour and hybrid cultivars from our collections were harvested at their optimum maturity and characterized according to their TPC, DPPH and ORAC values. RESULTS: The TPC content and DPPH and ORAC values varied greatly among the cherries, with the sour cultivars presenting higher levels than the sweet ones. The PCA plot showed a slight grouping by species and confirmed the high TPC content level in sour cultivars. The bi-colored cultivars had lower TPC and antioxidant capacity (AC) values than dark-colored ones, indicating that coloration could give an indication of the AC of fruits. No significant relationship between the agronomic and chemical properties was highlighted. CONCLUSIONS: Cherry fruits, especially from sour cultivars, represent an important source of bioactive compounds and could attract new interest as a ‘functional food’.

Summary: The aptitude for organogenesis from normal hormone-dependent cultures very commonly decreases as the tissues are serially subcultured. The reasons for the loss of regenerative ability may vary under different circumstances: genetic variation in the cell population, epigenetic changes, disappearance of an organogenesis-promoting substance, etc. The same reasons may be evoked for the progressive and eventually irreversible loss of organogenic totipotency in the course of neoplastic progressions from hormone-independent tumors and hyperhydric teratomas to cancers. As in animal cells, plant cells at the end of a neoplastic progression have probably undergone several independent genetic accidents with cumulative effects. They indeed are characterized by atypical biochemical cycles from which they are apparently unable to escape. The metabolic changes are probably not the primary defects that cause cancer, rather they may allow the cells to survive. How these changes, namely an oxidative stress, affect organogenesis is not known. The literature focuses on somatic mutations and epigenetic changes that cause aberrant regulation of cell cycle genes and their machinery

The present study aimed to assess antioxidant activities of three organs (flower, fruit, and stem) extracts of Tunisian Ferula (F.) communis. Various experimental models were used to characterize the antioxidant activities in vitro as well as on ROS-induced fluorescence using dichlorofluorescein technique from phorbol myristate acetate (PMA)-stimulated human myeloid cell line HL-60. Results showed that the antioxidant activities varied considerably with organs. Thus, flower exhibited higher DPPH-scavenging ability, reducing and chelating power than stem and fruit. Also, antioxidant capacities using ORAC method and a cell-based assay showed that fruit and stem exhibited statistically similar antioxidant activities. Moreover, F. communis contains high amounts of flavonoids with various health benefits attributed to their antioxidant potential. Likewise, to obtain biologically relevant information, the antioxidant activities of the extracts were evaluated on cellular models implicating the antioxidant activities; this test generally showed that F. communis flower extracts have the highest antioxidant capacities correlated to the highest total phenolic content. The identification of phenolic compounds in F. communis extracts using RP-HPLC revealed that resorcinol, ferulic, and syringic acids together with coumarin were the major molecules.

BACKGROUND: Small dark fruits represent one of the most important sources of bioactive compounds with antioxidant capacity in the human diet. The content of health-promoting antioxidants in these fruits may be important information to take into account when a fruit producer has to choose which cultivar to grow. OBJECTIVE: It is important to know how antioxidant capacity and antioxidant compounds as total phenolics and ascorbic acid vary between 9 small dark fruit species and for each species among cultivars (2 to 10 per species). METHODS: The antioxidant capacity (ORAC assay), total phenolic (Folin-Ciocalteu) and ascorbic acid content were measured in 9 fruits (plums, blackcurrants, blackberries, blueberries, cherries, redcurrants, raspberries, white currants and gooseberries) / 42 cultivars harvested at maturity during their high production period. RESULTS: The comparison of the average of the various cultivars of each small fruits showed that blackcurrants had the best antioxidant capacity (with plums), the highest ascorbic acid content and the highest total phenolic content (with blackberries). The present study shows that total phenolic compounds, ascorbic acid and antioxidant capacity strongly differed between genotypes of each small dark fruits. Other parameters as harvest time, culture conditions and maturity degree at the harvest may also influence the antioxidant capacity of small fruits. CONCLUSIONS: Among small dark fruits, blackcurrants have high qualities. Choices of variety, harvest time and maturity degree are important for all fruits.

Tubers can be initiated and developed in vitro from nodal cuttings of yam (Dioscorea cayenensis-D. rotundata complex). The effect of exogenous jasmonic acid, alone or in combination with putrescine, on these processes was investigated in relationship to endogenous jasmonic acid and polyamine levels. Application of exogenous jasmonic acid at various concentrations positively affected microtuber formation and growth from yam nodal cuttings. In control conditions, 3 weeks were needed to obtain 100% of tuberisation. Jasmonic acid at low level (0.1 μM) accelerated tuber formation (46% after 1 week) as did putrescine (10 μM). But endogenous levels of jasmonic acid were not significantly affected by its exogenous presence in the medium. Jasmonic acid also interacted with other growth regulators as polyamines, but the decrease in time necessary to observe tuber formation could not be correlated with endogenous modifications of PUT content. The presence of jasmonic acid (0.1–1 μM) as PUT (1 μM) induced also an increase of tuber length and weight. The combination of jasmonic acid (0.1 μM) and putrescine (1 μM) had no positive effect on tuber formation (precocity) but had an additive effect on further growth (length and weight). In the future, these results could help the optimising in vitro conditions for mass production of larger yam microtubers.

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01-10 microM MeJA, either under root-inductive conditions, i.e., on medium containing 10 microM indole-3-butyric acid (IBA) and 0.1 microM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1-4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 microM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 microM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.

Tuberisation was obtained in vitro on yam (Dioscorea cayenensis–Dioscorea rotundata complex). The effect of exogenous polyamines on tuber formation and development (length and weight of microtubers) was investigated and discussed in relation with changes in endogenous polyamines. Application of exogenous polyamines, inhibitors of their metabolism, and polyamines precursors in various concentrations positively affected microtuber formation by yam nodal cuttings and their further development. In control conditions, 3 wk are needed to obtain 100% of tuberisation. With low concentrations of putrescine (10−5 or 10−6 M), tuber formation occurred earlier. Polyamine endogenous level and metabolism can be significantly affected by exogenous polyamines, but modifications of endogenous free polyamines could not be directly correlated to the tuber formation process. Increases in endogenous putrescine and auxins were observed in tubers showing a better development in the presence of putrescine. These results can be used for optimising in vitro conditions for mass production of larger microtubers of the D. cayenensis–D. rotundata complex.

Apple rootstock MM106 shoots, raised in vitro, rooted at 96.7% after culture on a medium supplemented with an auxin for 5 d in darkness followed by culture on a second medium without growth regulators for 25 d in light. In control conditions (in absence of auxin in the first medium), these shoots did not root. Putrescine (PUT), spermidine (SPD), cyclohexylamine (CHA), and aminoguanidine (AG) enhanced rooting when applied during the first d of culture in the absence of IBA; on the contrary, α-difluoromethylornithine (DFMO) added to the first medium with IBA inhibited rooting. The endogenous levels of indole 3-acetic acid (IAA) and indole 3-acetylaspartic acid (IAAsp) increased up to a maximum concentration at days 2 and 3, respectively, in initial rooting conditions. PUT, when added with IBA, did not affect the typical IAA and IAAsp increase; when applied alone, it provoked an increase of their levels. Similar results were recorded with CHA. SPD, AG, and DFMO did not induce an increase of IAA and IAAsp in nonrooting conditions. The levels of endogenous PUT increased to a maximum at day 2 in rooting conditions; it was slightly affected by exogenous PUT and CHA application but reduced by SPD, AG, and DFMO. In rooting conditions, if the first medium was supplemented with SPD or AG, a small increase in peroxidase activity was observed, similar to that obtained with PUT treatment. The present work indicates an involvement of polyamines in the control of rooting and an interaction with auxins during the physiological phase of rooting. The consequence of this relationship was a different rooting expression, according especially to the content of these regulators in the culture medium.

The influence of lighting conditions, culture volume and four different auxins (indoleacetic acid (IAA), indolebutyric acid (IBA), 2,4-dichlorophenoxy- acetic acid (2,4-D) or 3-(benzo[b]selenyl)acetic acid (BSAA)) on Panax vietnamensis cell growth was evaluated in flasks. The highest biomass productivity was observed under continuous light and in the presence of IBA (0.36 g DW l−1 d−1) or BSAA (0.5 g DW|−1 d−1). Cultures in bioreactors were performed with these two auxins. The final biomass concentration was 107 g FW|−1 and 373 g FW|−1 in the presence of IBA or BSAA, respectively.

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.␣tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.

Lignification and xylem cell multiplication for wood formation were examined in in vitro propagated walnut shoot cuttings after transfer on an auxin- containing rooting medium for one week and subsequently during root development in vermiculite in the absence of growth regulators. Lignification in the shoot stems started immediately after the exogenous auxin treatment which implied changes in peroxidase activity and in free IAA levels. Sustained lignification required the completion of the following rooting phases. The lignin was exclusively located in xylem cells, the number of which increased with the number of developing roots. The mutual interactions between the aerial parts of the plants and their roots are discussed.

With the aim to differentiate the ionic and osmotic components of salt stress, short and long-term changes in free polyamines and proline induced by iso-osmotic concentrations of NaCl (0.1 mol/L and 0.2 mol/L) and mannitol (0.2 mol/L and 0.4 mol/L) were determined in Fraxinus angustifolia callus. The peculiarities of the short-term responses were: i) a very early (30 min) and temporary increase in Putrescine (Pu) and Spermine (Spm) as a consequence of salt treatment, and ii) a continuous accumulation of Spermidine (Spd) and Spm in response to mannitol. The changes of Proline (Pro) were quite limited both in the short and in the long term, and generally occurred later than Polyamine (PAs) changes took place, suggesting a regulatory mechanism of PAs metabolism on Pro biosynthesis. In the long-term, no drastic accumulations of Pro or PAs in response to NaCl and mannitol were observed, suggesting that their physiological role is unlikely to be that of osmo-compatible solutes in this plant system. The salt induced a higher callus growth inhibition effect than did mannitol and this inhibition was associated with the reduction of endogenous levels of PAs, especially Pu. However, while a diverging time course was observed under lethal salt concentration (0.2 mol/L NaCl), a high parallelism in the endogenous changes of Pro and Pu was observed under all non-lethal conditions (control--0.2 and 0.4 mol/L mannitol--0.1 mol/L NaCl). Therefore the synchronous changes of Pro and Pu can be considered as a physiological trait associated with cell survival. These results indicate a strong metabolic co-ordination between PAs and Pro pathways and suggest that the metabolic fluxes through these pathways start competing only when the stress level is high enough to be lethal for cells.

The lignin content of walnut shoots did not change during in vitro shoot multiplication. Lignin content started to increase as soon as shoots were passed to a rooting medium with auxin. Exogenous auxin (applied for rooting) caused a transient elevation of the endogenous free indoleacetic acid (IAA) content with a simultaneous decrease of peroxidase activity. These events typically marked the completion of the rooting inductive phase (before any visible histological event, that is before the cell divisions beginning the rooting initiation phase). This meant that either the given exogenous auxin or the endogenous IAA has served as signal for the stimulation of lignification. Continued increase of lignification in the shoots required completion of root formation; this increase indeed was slown down when root emergence did not occur. It was further shown that lignification varied conversely to the content of the soluble phenol content, itself apparently being related to the activity of phenylalanine ammonia-lyase activity.

The synthesis of 2,4-dichlorophenylselenoacetic acid (2,4-D-Se) may be completed in three steps starting from 2,4-dichloroaniline. The selenium is inserted in the molecule by reaction of a diazonium salt with potassium selenocyanate. 2,4-D-Se has been tested as an auxin in several bioassays including the regeneration of somatic embryos, adventitious root formation and the associated temporary increase of endogenous auxins at the induction phase, and callus formation, and compared with the natural auxin indoleacetic acid (IAA), the classical synthetic auxin(s) naphthaleneacetic acid (NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D), and with the synthetic seleniated IAA, 3-(benzo[b]selenienyl) acetic acid, BSAA. These biological assays classified 2,4-D-Se together with BSAA among the most powerful synthetic auxins. The role of selenium is briefly discussed.

The present research work describes the effects of 2,4-dichlorophenoxyacetic acid (2,4-d)/Benzylaminopurine (BAP) ratio on callus induction, total phenolic and ascorbic acid production, and antioxidant activities in leaf-derived calli of Crataegus azarolus (hawthorn). The supplementation of 1.0 mg/L 2,4-d and 1.0 mg/L BAP to MS medium was found to be the most efficient for callus induction (as percentage and fresh weigh). The results of biochemical analysis showed that the highest total phenolic contents were obtained in callus cultured on MS medium supplemented with 2.0 mg/L 2,4-d and 1.0 mg/L BAP (52 ± 0.56 mg GAE/g DM) and were significantly lower than those of intact leaves (76 ± 1.72 mg GAE/g DM). However, the highest ascorbic acid contents were found in callus cultured on MS medium supplemented with 1.0 mg/L 2,4-d and 0.5 mg/L BAP (0.96 ± 0.13 mg AAE/g DM) and these amounts were statistically similar to those found in leaf tissues (0.74 ± 0.07 mg AAE/g DM). Antioxidant activities of callus extracts were determined using two TEAC assays and results showed that extract of callus cultured on MS medium supplemented with 2.0 mg/L 2,4-d and 1.0 mg/L BAP have the greatest antiradical activities against DPPH (124 ± 2.92 mg TE/g DM) and ABTS (0.19 ± 0.02 mg TE/g DM) compared to the leaves of field-grown plant. Thus, the use of high level of 2,4-d over BAP can be suitable to enhance the quality more than the quantity of bioactive compounds in leaf callus culture of hawthorn.

Adventitious stipular bud formation occurred in vitro in many strawberry cultivars during the proliferation phase onmedium containing Knop macronutrients, MS micronutrients, vitamins, aminoacids,2.22 μM BAP, 2.46 μM IBA and 0.29μM GA3. As described previously for cultivarGorella,cultivar Elsanta also showed adventitious stipular buds developing on theabaxial median zone between the stipule tips. To compare the shoots producedfrom both types of buds, clonal propagation was initiated from stipular budsandfrom axillary buds on the above mentioned medium. Stipular buds were separatedfrom the meristem-tip initiated plantlet and cultivated in the presence of alower BAP concentration (1.33 μM) to prevent further stipularbud formation.

La regeneration in vitro de plants de Fraisier (Fragaria x ananassa Duch. cv. Elsanta) a partir de bourgeons adventifs stipulaires etait a l'origine de l'obtention d'un clone dont le comportement vegetatif et reproducteur in vivo s'est nettement distingue de celui de son homologue d'origine axillaire. Hormis la couleur claire et la forme arrondie de ses feuilles, ce nouveau clone se caracterise par un rythme plastochronal plus lent qui correspond aussi a la production d'un plus grand nombre de bourgeons axillaires sous forme de stolons. Au niveau floral, le receptacle est moins allonge et se trouve a la hauteur des etamines dont les antheres sont plus volumineuses que celles du clone axillaire. Ce dernier, dont le receptacle emerge au dela des etamines, presente une moins bonne pollinisation qui se traduit par une importante deformation des fruits, surtout en hiver et en l'absence d'agents pollinisateurs.

Because the term stress is used, most often subjectively, with variousmeanings, this paper first attempts to clarify the physiological definition,andthe appropriate terms as responses in different situations. The flexibility ofnormal metabolism allows the development of responses to environmental changeswhich fluctuate regularly and predictably over daily and seasonal cycles. Thusevery deviation of a factor from its optimum does not necessarily result instress. Stress begins with a constraint or with highly unpredictablefluctuations imposed on regular metabolic patterns that cause bodily injury,disease, or aberrant physiology. Stress is the altered physiological conditioncaused by factors that tend to alter an equilibrium. Strain is any physicaland/or chemical change produced by a stress, i.e. every established condition,which forces a system away from its thermodynamic optimal state. The papersecondly summarises the Strasser's state-change concept which is preciselythat suboptimality is the driving force for acclimation (genotype level) oradaptation (population level) to stress. The paper continues with the actualknowledge on the mechanisms of stress recognition and cell signalling. Briefly:plasma membranes are the sensors of environmental changes; phytohormones andsecond messengers are the transducers of information from membranes tometabolism; carbon balance is the master integrator of plant response; betwixtand between, some genes are expressed more strongly, whereas others arerepressed. Reactive oxygen species play key roles in up- and down-regulation ofmetabolism and structure. The paper shows finally that the above concepts canbeapplied to plant tissue cultures where the accumulating physiological andgenetical deviations (from a normal plant behaviour) are related to thestressing conditions of the in vitro culture media and ofthe confined environment. The hyperhydrated state of shoots and the cancerousstate of cells, both induced under conditions of stress in invitro cultures, are identified and detailed, because they perfectlyillustrate the stress-induced state-change concept. It is concluded that stressresponses include either pathologies or adaptive advantages. Stress may thuscontain both destructive and constructive elements : it is a selection factoraswell as a driving force for improved resistance and adaptive evolution.

The PR-10 protein family contains proteins related to pathogenesis and allergenic proteins. They are acidic proteins, with an intracellular localization, usually members of a multigene family and ubiquitous among plant kingdom. They contain a highly conserved motif, identified as a phosphate binding site. Even if the biological function of these proteins is still unknown, many indicators seem to favour the hypothesis of an RNAse activity. Moreover, one of them seems to be a cytokinin specific binding protein. Besides, PR-10 protein expression induction under cytokinin treatment was observed with the Catharanthus roseus CrPR-10a protein. Here, we present the evidence of a significative increase of endogenous cytokinin contents in leaves of transgenic Tobacco plants over-expressing the CrPR-10a protein.

The content of oxidized and reduced pyridine nucleotides and some enzymatic activities of the oxidative pentose phosphate and glycolytic pathways were compared in normal (NS, growing on agar) and hyperhydric (HS, growing on gelrite) shoots of Prunus avium L. after 4 weeks of in vitro culture. The chlorophyll fluorescence from leaves and the redox capacity of the plasma membrane (reduction of exogenously added ferricyanide) of both types of shoots were recorded. The pool of oxidized and reduced pyridine nucleotides was lower in HS than in NS. These results suggested a reduced metabolism of HS in comparison to normal ones. This hypothesis was also supported by other observations. First, chlorophyll fluorescence measurements showed a lower chlorophyll content and a slight reduction of the photosynthetic capacity in HS. Second, the low activity of some enzymes of oxidative pentose phosphate pathway (OPP) and glycolysis indicated a decline of these biochemical pathways in HS with the consequence of a reduced production of chemical energy in the form of NAD(P)H and ATP. Finally, the lower reduction of ferricyanide by HS suggested a lower rate of redox reactions at the level of the plasma membrane of these shoots in comparison to NS.

Node cultures of Eucalyptus camaldulensis Dehn inPetri dishes in vitro under darkness in the presence of anauxin developed meristematic agglomerates (4 to 6 diameter),i.e. dense shoot clusters in which outgrowth of numerous successive buds islimited. Similar cultures under a 16 photoperiod produced smallgreen plantlets with reduced leaves often presenting white hypertrophiedlenticels and very short roots crowning the stem bases. The use of half-litreglass vials under light allowed direct development of well-developed rootedplantlets, either in the presence of the same auxin or in the presence of acytokinin. Light favoured an increase in phenolic compounds and a reversevariation of peroxidase activity during the culture cycles. These aspects arediscussed in terms of a possible regulation of the endogenous auxin levelthrough a light control of peroxidase activity and the level of phenoliccompounds.

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the D8 wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurred at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delaye...

Summary In vitro propagation of cauliflower has generally been achieved through axillary shoot proliferation of curd explants on Murashige and Skoog (MS) medium supplemented with an auxin and a cytokinin. Recently, it has been shown (Vandemoortele 1999) that a soaking in sucrose (-2 MPa for 24 h) of cauliflower curd explants, before culture without any growth regulator, also induced axillary branching. The later procedure avoids the phenomenon of hyperhydricity in the shoots formed. Axillary shooting obtained by the two methods appears to be mediated by modifications of internal cytokinin levels. The osmotic pretreatment did not influence auxin levels, but induced a zeatin and a zeatin riboside levels increase. Curd explants cultured with the usual procedure (on MS medium supplemented with 5 μmol/L BA and 0.5 μmol/L NAA) showed a zeatin and zeatin riboside levels increase of the same magnitude and a higher one for isopentenyl adenine and isopentenyl adenosine. The modification of the cytokinin status in the curd explants subjected to a short osmotic pretreatment thus should be less favourable for hyperhydricity.

Eucalyptus camaldulensis can be micropropagated through so-called meristematic agglomerates (MAs). MAs (4–6 mm diameter) are dense shoot clusters initiated by the outgrowth of numerous successive buds. Their reddish nature is associated with an increase in their endogeneous cytokinin level during the exponential phase of growth. A simultaneous decrease in the auxin level favors a high cytokinin/auxin ratio. A low level of polyamines occurs at the time of the lowest level of auxins. Slow hormone release by activated charcoal plays a role in this very prolific organogenesis.

Summary In a fully habituated non-organogenic sugarbeet callus (HNO) overproducing polyamines and underproducing ethylene (in comparison with its normal hormone-dependent counterpart), the question raised about a possible competition between these two metabolites for their common precursor, S-adenosyl-methionine (SAM). The experimental strategy consisted in determining the effects of exogenous polyamines and inhibitors of polyamine biosynthetic pathway on growth, polyamine accumulation and ethylene production. Exogenous putrescine or spermidine decreased polyamine contents and ethylene production. Inhibitors of the diamine putrescine biosynthesis, DFMO and DFMA, induced a reduction of both polyamine content and ethylene production with an increase of HNO callus growth. However, when a mixture of the two inhibitors was used, an increase of ethylene production was observed without any effect on growth. The inhibitors of spermidine synthase (CHA) and of SAM decarboxylase (MGBG) also decreased polyamine content and ethylene production with different effects on growth according to the concentrations used. The combination of the two inhibitors (CHA + MGBG) increased ethylene production of the HNO callus. The effect of growth regulators (auxin and cytokinin) on growth and ethylene production of HNO callus is also discussed. These results suggest that polyamines affect directly the ethylene biosynthesis. In the absence of an exogenous hormonal control, the lower ethylene metabolism of HNO callus could not be explained by a competition with polyamines for their common precursor.

Rooting of wild-type tobacco (Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation.

The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, rac, of tobacco compared to its wild homologue. The mutant and wild shoots showed similar peroxidase variations along the growth cycle of 21 days, but with higher levels of activity for the rac mutant. During this growth cycle, the minimum of peroxidase activity occurred at day 14 for both tobacco whole shoots. However, this minimum of activity did not occur at the same day in the basal part of the stem, where roots may appear, of the two types of tobacco. Both mutant and wild whole shoots showed about the same IAA-oxidase activity in the fractions resulting from a gel filtration of the crude extracts through a Sephadex G-100 column but differed in the degree of auxin protection. The rac shoots exhibited a very high level of auxin protectors of low molecular weight, among which chlorogenic acid. They were also characterized by eight to nine times higher level of soluble phenolics. The relationships between these biochemical aspects in relation to the absence of root formation in the rac mutant are discussed.

A callus with embryogenic capacity was generated fromroot sections of Panax ginseng and used as aninoculum source for embryogenic liquid cultures in athree-step process: – a suspension culture of cellaggregates in the presence of an auxin/cytokininmixture, – an induction medium containing auxin only(for 5 to 30 days), – a regeneration medium containingcytokinin only (for one month). Up to 25 embryos wererecovered per 2.5 g of aggregates in these conditions.Incorporation of polyamines or their precursorsarginine and ornithine into either the induction orregeneration media increased the number of embryosproduced by up to 4 times. Inhibitors of bothbiosynthesis and biodegradation of polyamines reducedthe number of embryos. These results support earlierfindings of the role of polyamines in the process ofsomatic embryogenesis. The success of these liquidcultures opens up the possibility of producing somaticembryos of Panax ginseng in bioreactors.

A fully habituated (auxin- and cytokinin-independent) self-regenerating (organo-genic) sugar beet cell line (HO) and a fully habituated non-organogenic one (HNO) derived from the former one, were analyzed as to their nuclear size and DNA content. Flow cytometry and image analysis were used and cells of certified diploid leaves of the same sugar beet strain served as controls. The HNO cells had been shown previously to have many characteristics of cancerous cells. The analyses made on leaves and HNO cells indicated the presence of only one population of cycling cells. In HO cells, two cycling populations were detected: the first one had the same DNA content as the leaves while the second one contained two fold more DNA than the first population. HNO cells showed the higher nuclear size and DNA content. HNO cells also showed evidence of aneuploidy. Thus, nuclear size, DNA content and ploidy level increase together with the neoplasic progression to culminate in HNO cells with the loss of organogenic totipotency.

Lignosulfonates (LIGNs) are low-cost by-products from the paper industry and are already commercialized as fertilizers. Because earlier laboratory and glasshouse assays had shown a beneficial effect of LIGNs on rooting and general plant vigor, their incorporation in several plant tissue culture types was examined here. The present assays indicated that well-chosen concentrations of LIGNs, whether they were chelated with Ca or Fe, stimulated growth of a normal and an habituated sugarbeet callus, improved multiplication rate and vigor of a shoot-proliferating poplar cluster, and increased the rooting percentage of holly, ginseng, and poplar shoots. Complementing the exogenous rooting auxin with LIGNs enhanced the increases of endogenous levels of indoleacetic acid and its aspartate conjugate in the basal parts of poplar shoots at the rooting inductive phase. Although LIGNs exerted some effects in the absence of the growth regulators, they could not replace them. Their possible mode of action is discussed.

Summary Cytokinins (CKs) represent an important class of phytohormones particularly known for their antisenescence properties that might be regulated through an effect on the oxidative metabolism. In the present work, we demonstrate the effect of CKs on catalase activity in tobacco cultivated in vitro. The catalase activity observed in suspension-cultured cells decreased slightly during the first hour of CK treatment and increased thereafter to double the level detected in untreated cells. In contrast to these results, catalase activity was inhibited in shoot cultures in which the endogenous levels of CK were elevated by the introduction of the isopentenyltransferase gene or by an exogenous feeding of CK to the cultures. Interestingly, this catalase inhibition correlated with an accumulation of scopolin, an inducible coumarin. Taken together, our results show that CK modulates (directly or undirectly) catalase activity. The inverse relationship that was always found between scopolin accumulation and catalase activity is discussed in terms of vitrification and habituation.

Summary A fully-habituated and nonorganogenic (HNO) sugar beet callus was previously shown to overproduce polyamines, as compared with a normal (N) auxin- and cytokinin-dependent callus of the same strain. Because relationships were established between polyamine levels and metabolism with different growth and development processes, some key enzymes in the metabolic pathways of polyamines were investigated in the HNO callus, and their involvement in growth appraised. Putrescine was found to be the major free and conjugated polyamine in the HNO callus. It was biosynthesised preferentially via ornithine and ornithine decarboxylase (ODC), which is in agreement with the surplus of synthesised ornithine. Diamine (DAO) and polyamine (PAO)-oxidase activities were also highest in the HNO callus, as compared with the normal, with DAO being the more active. Transglutaminase activities(± Ca) were also higher in HNO than in normal callus. Addition of different polyamines or of inhibitors of their biosynthesis to the culture medium of the HNO callus modified the level of endogenous polyamines and affected callus growth. The results thus pointed out a higher polyamine metabolism, particularly of putrescine, in the actively growing auxin- and cytokinin-independent callus than in the normal one. They also provided evidence for the sensitivity of a habituated tissue type towards this class of growth regulators.

The paper describes a procedure for the initiation, subculture and continued proliferation of adventitious roots of Panax ginseng and Panax quinquefolium, which resemble hairy roots. The technique took advantage of the high powerful activity of a new synthetic auxin: benzo[b]selenienyl acetic acid (BSAA). Such initiation from root explants was dependent upon the season, the type and concentration of auxin. The hairy-like roots of ginseng could be subcultured by transfer every 4 weeks to fresh liquid medium either in agitated Erlenmeyer flasks or in bioreactors. Optimal conditions for a continued multiplication (up to 14 per month) were determined. The only practical problem was the limitation of the fresh mass as inoculum: the multiplication rate decreased with the increased quantity of roots. It is postulated that a root growth inhibiting substance was released into the media by the proliferating ginseng hairy roots.

A fully habituated (auxin- and cytokinin-independent) nonorganogenic (HNO) sugarbeet (Beta vulgaris) callus produces very little ethylene as compared with a normal (N) hormone-requiring callus of the same strain. Both callus types react by growth changes to application of inhibitors of ethylene biosynthesis and ethylene action, of 1-aminocyclopropane-1-carboxylic acid (ACC) as the immediate precursor of ethylene, to transfer from light to darkness, and also to application of exogenous ethylene or an ethylene trapper. This indicates their growth dependency upon their endogenously biosynthesized ethylene and also their sensitivity to exogenous gas. However, the sensitivity was generally higher for the HNO callus producing naturally less ethylene. The weaker reaction of the HNO callus to the exogenous ethylene was attributed to its hyperhydric status (a water layer surrounding the cells). Because low ethylene production appears as a general characteristic of habituated cell lines, the causal and/or consequential relationships of this low ethylene production with other characteristics of habituated tissues (absence of exogenous hormones in the culture media, deficiency of cell differentiation, accumulation of polyamines in neoplastic tissues) are discussed.

The interactions between cytokinins and ethylene on alkaloid accumulation in a periwinkle cell line have been examined. It was found that (a) either exogenously-applied cytokinins or ethylene (the latter through ethephon degradation) greatly enhanced ajmalicine accumulation in cells subcultured in a 2,4-dichlorophenoxyacetic acid-free medium; (b) the enhancing effect of cytokinin was not mediated by enhancement of endogenous ethylene production contrary to what is found in some plant models; (c) the responses to exogenous cytokinin and ethylene were additive and showed a different pattern of expression. It may be concluded that cytokinin and ethylene can up-regulate the alkaloid production in a periwinkle cells through independent pathways when added exogenously to the cultures.

Summary The amounts of some reductants (ascorbic acid, reduced glutathione, α-tocopherol) and the amounts of some markers of lipid peroxidation (peroxide and malondialdehyde) were quantified weekly in normal shoots (NS, in culture on agar) and in hyperhydrating shoots (HS, in culture on gelrite) of Prunus avium L. The redox activity of the plasma membrane (reduction of exogenously added ferricyanide), the antilipo-peroxidant potential, the level of hydrogen peroxide and the lipoxygenase (EC 1.13.11.12) activity were investigated after 28 days of culture in both types of shoots. Reducing capacity of HS seemed generally more efficient in comparison to NS: higher levels of free ascorbate, reduced glutathione and the antilipo-peroxidant potential were measured in HS than in NS. The α-tocopherol content did not change between the two types of shoots Reduction of exogenously applied ferricyanide was lower in HS during the last 2 weeks of the culture. These results suggest that the plasma membrane of HS had an unchanged reducing capacity but less redox transfer activity in comparison to NS. Markers of membrane damage (peroxide and malondialdehyde) were lower in HS than in NS and the same level of hydrogen peroxide was measured in the two types of shoots. Therefore, HS seem not to be submitted to oxidative stress. However, a more important lipoxygenase activity measured in HS was in contradiction to the lower peroxidation of lipids. The discussion points out some paradoxical results in an extensive classical analysis of stress criteria and indicates alternative defense mechanisms.

Summary To better understand the effect of habituation on gene expression in plant cells, we have compared the accumulation of specific mRNAs encoding respectively two proline-rich proteins, a chaperone protein and three enzymes linking primary and secondary metabolisms in two models of in vitro culture of periwinkle. These models consisted of two couples of a 2,4-dichlorophenoxyacetic acid-dependent/2,4-dichloro-phenoxyacetic acid independent line in which autonomy to auxin and cytokinin was obtained either through habituation or through transformation with the isopentenyltransferase gene from Agrobacterium tumefaciens . Results showed that gene expression was modified by plant growth regulator autonomy but differendy according to the type of autonomy: only the gene encoding a hydroxyproline-rich glycoprotein was regulated similarly in both PGR-independent lines. On the other hand, PGR autonomy did not lead to total insensitivity to exogenously-applied PGRs, and the two PGR autonomous lines did not accumulate indole alkaloids for different reasons.

Habituated sugar beet calli have been characterized as having a deficiency in some tetrapyrrole containing compounds. However, peroxidases might be dissociated from the other tetrapyrrole containing compounds. When light-cultured normal and habituated calli were transferred to darkness their peroxidase activity reduced and increased, respectively, indicating that habituation could not strictly be characterized by a deficiency in peroxidase content but rather by a different regulation of its activity. This regulation could be mediated through soluble effectors which act as potential peroxidase inhibitors and/or by a differential expression of the peroxidase isoenzyme patterns which were present in these tissues in both light and darkness. The different peroxidase activity and the nature of acidic and basic peroxidase isoenzymes in normal and habituated tissues could explain the different features of both types of cultures.

Shoots of the hybrid walnut Juglans nigra x Juglans regia contained serotonin in the micromole range and indole-3-acetic acid (IAA) in the nanomole range. The serotonin level fell by 40 % in 12 h in auxin (IBA) treated whole shoots and then reincreased to a maximum (50 %over the control) after 36 h. The same pattern was followed in the top portions of the shoot but in the shoot bases, serotonin always remained under the control level. The early decrease of serotonin was correlated with an increase in IAA-aspartate. The early decrease and peaking of the serotonin level preceded and corresponded to the increase and peaking of free IAA in the shoot bases. The initial serotonin pool in treated-to-root shoots might thus suffice for the biosynthesis of IAA and IAA-conjugated compounds. Because of its auxin-like properties, the early serotonin peak might be taken into consideration as an endogenous auxin signal for rooting in the present material. If this turns out to be so, the rooting signal for the shoot bases necessarily should come from the apices.

Microcuttings of seven genotypes of almond (Prunus dulcis Mill) used as peach rootstocks, showed different rooting ability in vitro. No direct relationship was found between peroxidase activity and total phenol content, determined in the whole microcuttings prior to the root inducing treatment, and the rooting ability of these genotypes. However, a positive relationship was found between free indole-3-acetic acid (IAA) level and IAA-aspartate and the rooting response. After transferring into the rooting medium, peroxidase activity of the easiest-to-root genotype increase up to a peak and then declined and the total phenol content showed an opposite trend. These variations did not occur in the most difficult-to-root genotype.

This is a short review of the classical and new, natural and synthetic plant hormones and growth regulators (phytohormones) and highlights some of their uses in plant tissue culture. Plant hormones rarely act alone, and for most processes— at least those that are observed at the organ level—many of these regulators have interacted in order to produce the final effect. The following substances are discussed: (a) Classical plant hormones (auxins, cytokinins, gibberellins, abscisic acid, ethylene and growth regulatory substances with similar biological effects. New, naturally occurring substances in these categories are still being discovered. At the same time, novel structurally related compounds are constantly being synthesized. There are also many new but chemically unrelated compounds with similar hormone-like activity being produced. A better knowledge of the uptake, transport, metabolism, and mode of action of phytohormones and the appearance of chemicals that inhibit synthesis, transport, and action of the native plant hormones has increased our knowledge of the role of these hormones in growth and development. (b) More recently discovered natural growth substances that have phytohormonal-like regulatory roles (polyamines, oligosaccharins, salicylates, jasmonates, sterols, brassinosteroids, dehydrodiconiferyl alcohol glucosides, turgorins, systemin, unrelated natural stimulators and inhibitors), as well as myoinositol. Many of these growth active substances have not yet been examined in relation to growth and organized developmentin vitro.

Fully habituated organogenic and nonorganogenic sugarbeet calluses reacted to application of the synthetic auxin [3-benzo(b) selenienyl] acetic acid by changes in growth and ethylene production. Treatment of fully habituated cells of periwinkle with 2,4-dichlorophenoxyacetic acid led to the decrease of free cytokinin contents (isopentenyl adenine, zeatin riboside, and zeatin) during the late exponential phase of growth. The polyamine contents were also modified and the capacity to biotransform secologanin into ajmalicine was decreased. Treatment of the habituated periwinkle cells with zeatin greatly increased the amount of a polypeptide of 16 kDa; this response was more marked than that displayed by the auxin-dependent line. These data show that hormone-independent calluses and cell suspensions can retain some sensitivity to growth hormones. However, differences of responses were observed between the auxin-dependent lines and the habituated lines.

Changes in endogenous free IAA and its conjugate IAAaspartate as well as in endogenous polyamines were analysed in in vitro poplar shoots during their inductive phase, under the effect of the rooting auxin NAA, in combination or not with polyamines or inhibitors of polyamine biosynthesis. In vitro raised poplar shoots rooted 100% when treated by NAA (0.3 mg/l) for 7 h, the previously determined duration of the inductive phase. Spermidine and aminoguanidine (AG, an inhibitor of diamine oxidase) alone were unable to promote rooting under these conditions, and counteracted the NAA rooting effect. Putrescine and cyclohexylamine (CHA, an inhibitor of spermidine synthase) on the contrary did not oppose the NAA effect and promoted up to 40% rooting when applied alone. The levels of free indoleacetic acid (IAA) and of its aspartate conjugate IAAsp elevated up to peaks situated at the 7th and 8th h, respectively in the basal parts of the NAA-treated shoots. Putrescine, when added toegether with NAA, did not affect the typical IAA and IAAsp increases (except a displacement of the peaks to the 8th and 10th h, respectively); when applied alone, it provoked elevation of their levels to limited extents. Quite similar results were recorded with CHA. Spermidine and AG counteracted the NAA induced elevations of IAA and IAAsp. The level of endogenous putrescine typically increased up to a peak at the 6th h in the basal part of the NAA-treated shoots only, was slightly affected by exogenous putrescine and CHA application but significantly reduced by spermidine and AG supply. The NAA inducing rooting treatment did not affect the variation of the levels of endogenous spermidine and spermine but putrescine treatment (also spermidine to a lesser extent) led to increases of their levels. The relationships between auxin and putrescine temporary accumulation are discussed as well as the involvement of the different polyamines in the rooting inductive process.

Summary Poplar shoots raised in vitro rooted by 100 °lo in the presence of an auxin while in its absence they did not root. Putrescine, however, and the inhibitor of spermidine synthase, cyclohexylamine (CHA), which favours putrescine accumulation, were able to promote up to 40 % rooting in the absence of auxin. The inhibitory effect of aminoguanidine (AG), which inhibits diamine oxidase, indicated that the catabolic pathway of putrescine was involved in the rooting process. The inductive phase of rooting in poplar shoots was characterized by an increase of peroxidase activity followed by a rapid decrease. Putrescine and CHA favoured this variation. AG and spermidine had adversary effects.

Vitrification of shoots of Prunus avium L. L. was induced and expressed in a four week in vitro multiplication cycle simply by replacing agar by gelrite. The first vitrification symptoms were visible from the 7th day on. Enzymatic antioxidants were compared weekly in crude extract of normal (on agar) and vitrifying (on gelrite) shoots. The activity of superoxide dismutase was higher in vitrifying shoots. The other enzymes (gaiacol-peroxidase, catalase, ascorbate peroxidase, mono- and dehydro-ascorbate reductases, glutathione reductase) had lower activities. Increased superoxide dismutase activity might mean hydrogen peroxide accumulation and decreased activities of the other enzymes, deficiency in its detoxification. The question therefore is raised whether the hyperhydric morphological abnormalities result from the accumulation of toxic oxygen forms. Vitrification is often considered as a morphological response to several stresses. Contrary to most plants which adapt themselves to stresses by increasing all the above defence enzymes, in vitro shoots under vitrifying conditions appear unable to react in a similar manner.

Strawberry plants issued from shoots raised after a high number of subcultures (HNS) in vitro, which were previously characterized by an abnormal hyperflowering process ex vitro, were multiplied conventionally through runners for 8 generations, and then meristem tips re-entered in vitro for a low number of shoot subcultures (LNS). Such HNS-LNS vitroplants flowered abnormally abundantly as compared with always conventionally propagated N-LNS vitroplants, thus self-perpetuating a long term memory of an abnormal developmental pattern. The HNS-LNS hyperflowering vitroplants were also characterized by a carry-over of other biological (reduced growth habit), morphological (reduced hairiness and deposition of epicuticular wax, unifoliolate leaves prevailing) and biochemical (higher peroxidase activity) traits. They also proliferated in vitro at higher rates, which corresponded to higher endogenous levels of cytokinins and higher cytokinin/auxing ratios. Unifoliolate leaves and high cytokinin/auxin ratios are recognized criteria of rejuvenation. These characteristics of HNS strawberry vitroplants are carried-over in the next generations through heritable gene expression.

Micropropagated poplar shoots rooted 100% on a rooting medium (A) containing NAA, but they did not root in the absence of auxin (NA). Putrescine, but not spermidine and spermine, promoted rooting up to 42% when added to the NA medium. Cyclohexylamine (CHA), an inhibitor of spermine synthase, also promoted (up to 36%) rooting in the absence of auxin. The inhibitors of polyamine biosynthesis DFMA (α-difluoromethylarginine) and DFMO (α-difluoromethylomithine), aminoguanidine (AG) and methylglyoxal-bis-guanylhydrazone (MGBG), inhibited rooting when applied in the presence of auxin and had no effect in its absence. The rooting inductive phase (in the presence of auxin) was determined by periodical transfer of shoots from A to NA medium, and by changes in peroxidase activity, to be 7 h. Putrescine (not spermidine and spermine) accumulated to a maximum during the inductive phase. Both putrescine and CHA promoted rooting on NA medium when applied during the first 7 h. In contrast DFMA and AG inhibited rooting during this period. The results point to the involvement of putrescine and its Δ1-pyrroline pathway, in the inductive phase of rooting in poplar shoots.

Summary Compared with a normal (N) auxin- and cytokinin-requiring sugarbeet callus, two habituated (a nonorganogenic HNO and a self-shooting HO) calli, obtained from the same mother strain produced a very low amount of ethylene. Ethylene production by HO callus cells in the presence of 1-aminocyclopropane-1-carboxylic acid (ACC) was increased by about 20 times versus 3-fold for HNO cells. The polyamine content of HNO cells was very high compared with N cells, but that of HO was lower. The results are compared with literature data on hormone autonomous lines and shooty tumors. The possible interactions between ethylene and polyamine metabolism are also discussed.