Dark-induced changes in the content of phenolic acids in callus culture of alfalfa (Medicago sativa)

The changes in the content of free and esterified phenolic acids extracted with methanol and the phenolic acids nonextractable with this solvent were studied during a long term cultivation (4 subcultures, 27 weeks total) of alfalfa callus in darkness and light. The total content of phenolic acids in the callus grown in darkness was lower as compared to their content in callus cultivated under 10/14 h light/darkness regime. It was decreasing during cultivation differently in the two treatments being in light-grown callus dependent on the length of cultivation and decreasing by each subcultivation in darkness. The portion of benzoic acid derivatives related to the total phenolic acids changed with the age of the culture reaching 90 % after the fourth subcultivation in darkness while only 45 % in light. Considerable decrease of the content of esterified form of phenolic acids accompanied by relatively stable content of free and firmly bound phenolic acids caused significant decrease of esters as related to total phenolic acids (from 87 % to 25 %).     

Phenolic substances in tissue cultures ofCentaurium erythraea

Phenolic compounds l,2,3-trihydroxy-5-methoxyxanthone, l-hydroxy-3,5,6,7, 8-pentamethoxyxanthone, and l,8-dihydroxy-2,3,4,6-tetramethoxyxanthone predominate in the callus tissue ofCentaurium erythraea, their content increasing with culture age. By contrast, the contents of the derivatives of cinnamic, chlorogenic, and ferulic acids decrease or do not change. In the cell suspension culture ofC. erythraea a larger amount of xanthones is synthesized than in the callus from which the suspension culture was derived. The content of phenolic acids is lower in the suspension culture than in the callus, but a larger number of low-molecular-mass phenolic substances occurs in the suspension culture than in the callus tissue.     

Role of phytohormones in organogenic ability of elm multiplicated shoots

The study presents the comparative analyses of endogenous contents of auxin (IAA), cytokinins (CKs), polyamines (PAs), and phenolic acids (PhAs) in apical and basal parts of elm multiplicated shoots with regard to the organogenic potential. The shoot-forming capacity was higher in the apical part than in the basal part. However, the timing of root formation was in the apical type of explant significantly delayed (compared with the organogenic potential of basal part). Significantly higher contents of free bases, ribosides and ribotides of isopentenyl adenine, zeatin and dihydrozeatin that were found in the apical segments, might be considered as the most important factor affecting in vitro shoot formation. The content of endogenous free IAA was approximately three times higher in the basal shoot parts than in the apical parts. The amounts of putrescine and spermidine were higher in the apical part which generally contains less differentiated tissues than the basal part of shoot. The predominant PhA in both types of explants was caffeic acid, and concentrations of other PhAs decreased in the following order: p-coumaric, ferulic, sinapic, vanillic, chlorogenic, p-hydroxybenzoic and gallic acids. The contents of all determined PhAs in their free forms and higher contents of glycoside-bound p-coumaric, ferulic and sinapic acids, precursors for lignin biosynthesis, were found in the basal parts.     

Effect of light quality and tissue origin on phenolic compound accumulation and antioxidant activity in Camellia japonica calli

In Camellia japonica callus culture, the effects of light quality on the accumulation of phenolic compounds were examined. To this end, the calli were cultured under three monochrome [white (W), red (R), and blue (B)] and two mixed [red/blue (RB) and red/green/blue (RGB)] light sources. After 4 weeks of culture, fresh and dry callus weights were determined, and phenolic content was analyzed using high-performance liquid chromatography. We also examined the effects of RGB light on calli derived from four origins (leaf: CL; root: CR; petal: CP; and ovary: CO). Total phenolic and flavonoid content and DPPH radical scavenging activity were highest in calli exposed to RB and RGB. Catechin was detected only in calli exposed to mixed light (RB and RGB). Interestingly, red pigmentation development in CL and CR calli after 2 weeks of culture and organogenic calli in CR and CP samples was observed. Furthermore, expression of the phenylalanine ammonia lyase and chalcone synthase genes was found to be higher in the CR line than in other lines, and total phenolic and flavonoid content was also higher in the CR and CP lines. The findings of the present study revealed that the accumulation of phenolics and flavonoids in callus tissue varies depending on light quality, which stimulates cell division, and may thus affect the proliferation and organogenesis of C. japonica callus. We also established that the effects of light on red pigment expression and the accumulation of bioactive compounds are dependent on the tissue origin of callus material.

     

Changes in the content of phenolic substances during the growth ofNicotiana tabacum cell suspension culture

The dynamics of changes in the content of four groups of phenolic substances was investigated during the growth cycle of the cell suspension culture ofNicotiana tábacum by means of fractionation. The relative contents of free phenolic acids, their esters, phenolic glycosides, and phenole acids non-extractable with methanol changed in dependence on the growth phase of the culture. A sharp increase, especially in the content of ester- and glycoside-bound phenolics and to a lesser extent also of phenolics belonging to the other two groups, occurred at the end of the lag phase. Then, after a temporary decrease at the early linear phase, the level of phenolics in the three fractions representing bound forms considerably increased again at the late linear and early stationary phases. The synthesized phenolic substances were partially released from the cells into the cultivation medium, which contained 15 to 30 % of the total content of the phenolics in the culture at different phases of the growth cycle. Likely causes of these changes are discussed.     

Involvement of phenolic acids in disease resistance of potato tubers from CEPA-treated plants

Treatment of vegetative parts of potato plants two weeks before the harvest with 0.2% 2-chloroethylphosphonic acid (CEPA) delayed the sprouting of tubers and increased the resistance of tubers to infections caused byPhytophthora infestans, Erwinia carotovora andFusarium spp. during the storage period. Levels of free, soluble ester- and glycoside-bound phenolic acids and cell wall-bound phenolics were determined in cortical parenchyma of tubers (periderm). The enhancement of phenolic acids in tubers from treated plants was caused primarily by the increase in the contents of free vanillic, caffeic andp-hydroxybenzoic acids and cell wall-bound ferulic, vanillic andp-coumaric acids.     

Haltbarkeit von Silagen und die sie beeinflussenden Faktoren

Content of p‐coumaric (PCA) and ferulic (FA) acid was determined by the HPLC method in fourteen forbs with a potential utilization as forages (range of nutrient content per kg DM: 100 to 244gCP, 339 to 528 g NDF and 180–369 g ADF. PCA and FA were determined after methanol extraction in four fractions: free phenolic acids extracted into ether, ester‐bound phenolic acids after alkaline hydrolysis, glycoside‐bound phenolic acids after acid hydrolysis, and cell wall‐bound phenolic acids after alkaline hydrolysis of the solid residue after the extraction with methanol.

Cell wall‐bound phenols were quantitatively the most important fraction (50% of total PCA and 47% of total FA, respectively). The differences among plant species in total PCA plus FA content were significant (F‐value 775, P < 0.01). The range of total phenol content was 31.3 to 416.3 mg/100g DM, the overall mean was 84 mg/100g DM.

Content of phenolic acids was correlated neither with ADF, NDF or ADL content (R ² = 1–3%, P > 0.05) nor with CP degradability (R ² = 3% and R ² = 1% for PCA and FA, respectively, P > 0.05).

95.4% and 30.9% of total PCA, and 98.3% and 72.5% of total FA disappeared in the rumen from the sample of Glechoma hederacea (species with the highest phenol content) and from the sample of Galium aparine (species with low phenol content), respectively, within the four hour incubation interval.

It is presumed that in comparison with grasses, PCA and FA concentration in tested forbs represents a much lower risk in potential ruminant nutrition.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Properties of Rice Straw Extract after Subcritical Water Treatment

Rice straw was separated into four parts: the upper, middle, and lower parts of the stem, and the leaf. They were treated with subcritical water at 140 to 260 °C. The yield, carbohydrate, protein, and phenolic contents were obtained as well as the UV-Vis absorption spectra and the radical scavenging activity of the extracts. The extracts obtained from the stem parts had almost the same characteristics and were different from those of the leaf part. The extracts, prepared at higher temperature, exhibited higher radical scavenging ability. The radical scavenging ability and the phenolic content showed a correlation (R ²=0.92), suggesting that the phenolic substances in the extract cause its antioxidant ability.     

Metabolism of phenolic compounds in <Emphasis Type="Italic">Vitis riparia</Emphasis> seeds during stratification and during germination under optimal and low temperature stress conditions

We studied the alterations in phenolic compounds in grape seeds during their stratification and germination under optimal conditions (+25 °C) and at low temperature (+10 °C). Biological materials in the study were seeds of Vitis riparia. Phenolic compounds were extracted from defatted seeds using 80 % methanol or 80 % acetone. The content of total phenolics was determined with the Folin-Ciocalteau reagent, while the content of tannins was determined by vanillin assay and the protein (BSA) precipitation method. The RP-HPLC method was used to determine phenolic compounds (phenolic acids, catechins) in the extracts. High amounts of tannins, catechins, gallic acid and lesser amounts of p-coumaric acid were found in the seeds. The content of total phenolics in acetone extracts was higher than that obtained using methanol. The amounts of phenolic acids and tannins found in V. riparia seeds after stratification were much lower. It may confirm a possible role of these compounds in dormancy of V. riparia seeds. After 72 h of low temperature treatment, inhibition of grape root growth and biochemical changes in seeds were detected. The chilling stimulated increased accumulation of some phenolic compounds (free gallic acid and catechins) in the seeds. These substances can protect plants against some abiotic stressors.     

Alternations in phenolic acids content in developing rye grains in normal environment and during enforced dehydration

A series of high pressure liquid chroamtography analyses revealed the presence of five phenolic acids in rye caryopses (vanillic, caffeic, p-coumaric, ferulic and sinapic), three of which (p-coumaric, ferulic and sinapic) were found in the free phenolic fraction. Ferulic acid was predominant, both among free acids and total phenolic acids (i.e. free, liberated from soluble esters and glycosides). The highest content of the free phenolic acids in rye caryopses was observed at the beginning of development, when on 22 DAF it was estimated at 11.55 μg·g⁻¹ DW. During dehydratation the total level of free phenolic acids in rye caryopses decreased in all investigated samples. Although total phenolic acids contents in all samples of unripe rye caryopses always decreased after dehydration, in rye sample collected in full ripeness (57 DAF), the amount of these compounds increased after the enforced dehydration. It should be added that in ester-bound-soluble phenolic acids fraction (the largest part in the total phenolic acids fraction), irrespective of the total amount decrease, much increase of sinapic acid content in this fraction was observed after dehydratation treatment in all investigated samples of caryopses of various ripeness. During the development and ripening of rye caryopses, a gradual increase in the precocious germination ability of the grain was observed. The enforced dehydration stimulated the process of precocious germination of developing and ripening rye caryopses. A possible role of phenolics in preventing precocious germination and acclimation to dehydration of developing and ripening rye grains is discussed.     

Caffeic acid derivatives from a hairy root culture of Lactuca virosa

In a search for biologically active phenolics, a hydroalcoholic extract from the hairy roots of Lactuca virosa was fractionated by chromatographical methods. The procedure led to the isolation of a substantial amount of 3,5-dicaffeoylquinic acid (3,5-DCQA)—a potent free radical scavenger. An analytical RP-HPLC separation of the hydroalcoholic extract from the hairy roots allowed identification of further hydroxycinnamates: caftaric acid (CTA), chlorogenic acid (5-CQA) and cichoric acid (DCTA), as well as small amounts of unbound phenolic acids. A time course of growth and caffeic acid derivatives accumulation in the hairy root culture was also investigated. The highest contents of the compounds in the examined roots were detected at the logarithmic phase of growth. The average content of 3,5-DCQA in the roots (ca. 2.5% DW) was at least one order of magnitude higher than that found in roots of Lactuca species and callus culture of L. virosa.     

Biological effects of water-soluble soil phenol and soil humic extracts on plant systems

The aim of this research was to extract, with a sequential method, HS free from water-soluble phenols (HS-WP) and water-soluble phenols free from humic substances (WP-HS), and to evaluate and to compare the biological effects of these two extracts to humic substances (HS) and water-soluble phenols (WP) traditionally extracted. In each extract we determined the concentration of low molecular weight organic acids, soluble carbohydrates, fatty acids, phenolic acids and total proteins. We tested the biological activity of each soil extract and of the single identified compounds on different plant organs (Pinus laricio callus, Daucus carota cells, and Pinus laricio, Pinus halepensis, Lens culinaris and Cichorium intybus seeds). The results showed that eliminating from HS the WP fraction, and from WP the HS fraction we obtained extracts chemically different from HS as such, and WP as such. HS and HS-WP increased callus and cell growth and also root elongation of the different species used; in contrast, WP and WP-HS had negative effects inhibiting callus and cell growth and seed germination percentage of coniferous, herbaceous and leguminous species. The negative effects can be ascribed to the presence of phenolic acids in the extracts while the positive biological activity can be attributed to the presence of tartaric acid, and fatty acids. In conclusion, this study helps to discriminate the effects of humic substances against phenolic compounds extracted from soils explaining the different and in some way contradictory biological behaviour of these two main fractions of SOM.     

Influence of some exogenous amino acids on the production of maize embryogenic callus and on endogenous amino acid content

The effects of four exogenous amino acids (proline, glycine, asparagine and serine) on the production of maize embryogenic callus and on its endogenous amino acid content have been investigated. For this purpose, an established embryogenic line of Type 1 callus from the inbred W64Ao2 has been used. From the results it may be concluded that a concentration of proline exceeding 6 mM is negative for the production of embryogenic callus. When proline is eliminated from the medium, other amino acids tested in certain concentrations yield a percentage of embryogenic callus production that exceeds or equals that of proline. The endogenous free proline content in embryogenic callus is significantly higher than that in non-embryogenic callus regardless of proline presence in the medium. The only exception are the glycine-containing media, in which endogenous free alanine of embryogenic callus increases at the expense of endogenous free proline. This study suggest a positive role of endogenous free proline or alanine accumulation in the embryogenic callus production which might be related to an adaptation to the metabolic changes produced by in vitro culture and embryogenesis induction. Furthermore, these results indicate that treatments with amino acids that are different from proline can be used to improve the efficiency of embryogenic callus production from well established maize callus cultures.Abbreviations Ala alanine - Asn asparagine - 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic callus - nEC non-embryogenic callus - Gaba gamma-aminobutyric acid - Glu glutamic acid - Gly glycine - Pro proline - Ser serine     

Composition of cell wall phenolics and polysaccharides of the potential bioenergy crop –Miscanthus

The composition and concentrations of cell wall polysaccharides and phenolic compounds were analyzed in mature stems of several Miscanthus genotypes, in comparison with switchgrass and reed (Arundo donax), and biomass characteristics were correlated with cell wall saccharification efficiency. The highest cellulose content was found in cell walls of M. sinensis‘Grosse Fontaine’ (55%) and in A. donax (47%) and lowest (about 32%) in M. sinensis‘Adagio’. There was little variation in lignin contents across M. sinensis samples (all about 22–24% of cell wall), however, Miscanthus×giganteus (M × g) cell walls contained about 28% lignin, reed – 23% and switchgrass – 26%. The highest ratios of cellulose/lignin and cellulose/xylan were in M. sinensis‘Grosse Fontaine’ across all samples tested. About the same total content of ester‐bound phenolics was found in different Miscanthus genotypes (23–27 μg/mg cell wall), while reed cell walls contained 17 μg/mg cell wall and switchgrass contained a lower amount of ester‐bound phenolics, about 15 μg/mg cell wall. Coumaric acid was a major phenolic compound ester‐bound to cell walls in plants analyzed and the ratio of coumaric acid/ferulic acid varied from 2.1 to 4.3, with the highest ratio being in M × g samples. Concentration of ether‐bound hydroxycinnamic acids varied greatly (about two‐three‐fold) within Miscanthus genotypes and was also the highest in M × g cell walls, but at a concentration lower than ester‐bound hydroxycinnamic acids. We identified four different forms of diferulic acid esters bound to Miscanthus cell walls and their concentration and proportion varied in genotypes analyzed with the 5‐5‐coupled dimer being the predominant type of diferulate in most samples tested. The contents of lignin and ether‐bound phenolics in the cell wall were the major determinants of the biomass degradation caused by enzymatic hydrolysis.     

Studies on Nitrogen Metabolism in Tobacco Plants A

The effect of age on non-protein constituents of tobacco leaves (N. tabacum L., var. Bright Yellow) has been studied. For this purpose leaves in three different stalk positions, upper, middle and lower, which represent young, mature and over-mature leaves, respectively, were harvested in the day-time and at night.

The total amino nitrogen content both in the day-time and at night decreases from the upper leaf position downwards. As for the individual groups, the content of both upper and middle leaves increases in the day-time and that of the lower ones increases at night.

In general, the content of the individual amino acids is high in the upper leaves and low in the lower ones. Proline and γ-aminobutyric acid, as a ratio of the total amino acid content, show a marked difference with position, in other words with age of the leaves.

The levels of proline decrease very sharply from the upper leaf position downwards and that of γ-aminobutyric acid exhibits an opposite trend in both samples at night and in the day-time. These trends are very prominent in the case of the midribs.

The contents of other amino acids, regardless of position, show similar trends with time to those reported in the previous paper₁₎, and the aspartic acid content increases at night.     

Melatonin influence on in vitro callus induction and phenolic compound production in sweet basil (Ocimum basilicum L.)

The aim of the present study was to evaluate melatonin effects on the callus induction and phenolic compound production of Ocimum basilicum L. (sweet basil). Calluses, derived from leaf explants, were grown on Murashige and Skoog (MS) medium supplemented with 0, 100, or 200 μM melatonin, and subsequently extracted for determination of their phenolic contents. Melatonin decreased the callus induction in both concentrations. Based on the phytochemical analysis, the highest total phenolic acid contents (784.6 μg g⁻¹ and 335.2 μg g⁻¹, respectively) were recorded in calluses grown in 100 and 200 μM melatonin-supplemented medium, compared with the calluses induced with MS alone (192.0 μg g⁻¹). Among the five phenolic acids confirmed in the callus samples, rosmarinic acid was the major constituent. The amount of rosmarinic acid increased significantly in callus grown on 100 μM melatonin medium by nearly 5-fold (754.2 μg g⁻¹), compared with the control group callus. Major volatiles in basil calluses were represented by 3-methylbutanal, benzaldehyde, 1,8-cineole, 2-nonenal, eugenol, and methyl eugenol, and these were in the ranges of 4 to 14%, 24 to 50%, 2 to 3%, 0 to 0.55%, and 2 to 17% (in relative percentages), respectively. The qualitative and quantitative analyses of these substances found in calluses formed on melatonin-supplemented or melatonin-free medium were evaluated separately.

     

PEG胁迫对不同培养方式下金发草愈伤组织再生能力的影响

在静置液体培养和振荡液体培养方式下,研究了聚乙二醇(PEG)胁迫对金发草[Pogona the rumpaniceum(Lam.)Hack]愈伤组织的再生能力和游离脯氨酸(F-Pro)积累的影响。发现金发草愈伤组织具有较高的耐PEG胁迫能力,培养方式与PEG胁迫对其再生抑制上存在时间与程度两个方面的表现。振荡培养方式主要表现为延迟再生时间,而PEG胁迫则主要表现为降低再生频率。两种培养方式都能使愈伤组织在PEG胁迫下发生F-Pro积累,F-Pro含量随PEG浓度的增加和培养时间的增长而升高。去胁迫后,大部分愈伤组织都能够恢复再生能力,F-Pro可能在抑制和恢复过程中作用较复杂。因此,在利用愈伤组织筛选抗旱植株时,PEG浓度应在300g.L-1以上,处理时间3周以上,静置培养更有利于抗旱突变体的筛选。     

In Vitro Selection for Salt Tolerance in Rice

In six cultivars of rice (Oryza sativa L.), Pusa Basmati 1, Basmati 370, Type III, Pant Dhan 4, CSR 10 and Pokkali, embryogenic callus growth, plant regeneration, and proline and total protein contents were studied under salt stress (on agar solidified media containing 0, 0.5, 1.0, 1.5 and 2.0 % NaCl). Four weeks after inoculation the callus fresh mass decreased with increasing salt concentration in all the six cultivars. The regeneration frequency in salt stressed callus was also lower as compared to control. 15 d and 30 d after inoculation proline content increased several fold whereas total protein content decreased markedly with increase in salt concentration.     

Effect of Plant Growth-Promoting Rhizobacteria and Culture Filtrate of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> on Phenolic and Salicylic Acid Contents in Chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea (Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident. Received: 1 April 2002 / Accepted: 4 May 2002