Effect of nitrate supply on the in-vivo synthesis and distribution of trifollin A,a Rhizobium trifolii-binding lectin,in Trifolium repens seedlings

In-vivo synthesis of the white-clover lectin, trifoliin A, was examined by the incorporation of labeled amino acids into protein during heterotrophic growth of intact Trifolium repens L. seedlings. Lectin synthesis was quantified by measuring the level of labeled protein immunoprecipitated from root exudate, from the hapten (2-deoxyglucose) eluate of the roots, and from root and shoot homogenates. The presence of labeled trifoliin A was confirmed by non-denaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography and comparison with trifoliin A standards. In-vivo-labeled trifoliin A was detected in seedling root homogenate 2 h after the addition of labeled amino acids and on the root surface by 8 h. Incorporation of labeled amino acids into protein and trifoliin A was greatest with 2-d-old seedlings and was greater when the plants were grown continuously in the dark than when they were exposed to 14 h light daily. Significantly more labeled lectin accumulated on the root surface of seedlings grown with 1.5 mM KNO₃ than of seedlings grown either without N or with 15.0 mM KNO₃. The labeled lectin from the root surface in all nitrate treatments and from the rootexudate samples of seedlings grown N-free and with 1.5 mM KNO₃ was fully able to bind to Rhizobium trifolii. In contrast, only 2% of the immunoprecipitable protein found in the root exudate of seedlings grown with 15.0 mM KNO₃ was able to bind to the bacteria. Thus, excess nitrate does not repress the synthesis of trifoliin A in the root, but does affect the distribution and activity of this newly synthesized lectin in a way which reduces its ability to interact with R. trifolii. By using Western blot analysis, much more total trifoliin A is detected in the homogenates of shoots than roots. However, greater than 80% of the total labeled protein and 85–90% of the total labeled lectin were found in the root homogenates of 2-d-old dark-grown seedlings incubated for 5 h with labeled amino acids. In addition, Western blot analysis indicated that the shoot homogenate contained smaller-molecular-weight peptides which reacted with the specific anti-trifoliin A antibody. These studies indicate that stored trifoliin A in the seed is degraded in the shoots during seedling development, while newly synthesized trifoliin A in the roots is excreted to the root surface and external environment.Abbreviations IgG immunoglobulin G - LPS lipopolysaccharide - PBS 10 mM potassium-phosphate buffer, pH 7.0, containing 0.8% NaCl - PBS-T 20 mM phosphate-buffered saline, pH 7.4, containing 0.05% Tween 20 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Counteraction of Salinity Stress on Wheat Plants by Grain Soaking in Ascorbic Acid, Thiamin or Sodium Salicylate

The interactive effects of salinity stress (40, 80, 120 and 160 mM NaCl) and ascorbic acid (0.6 mM), thiamin (0.3 mM) or sodium salicylate (0.6 mM) were studied in wheat (Triticum aestivum L.). The contents of cellulose, lignin of either shoots or roots, pectin of root and soluble sugars of shoots were lowered with the rise of NaCl concentration. On the other hand, the contents of hemicellulose and soluble sugars of roots, starch and soluble proteins of shoots, proline of either shoots or roots, and amino acids of roots were raised. Also, increasing NaCl concentration in the culture media increased Na⁺ and Ca²⁺ accumulation and gradually lowered K⁺ and Mg²⁺ concentration in different organs of wheat plant. Grain soaking in ascorbic acid, thiamin or sodium salicylate could counteract the adverse effects of NaCl salinity on the seedlings of wheat plant by suppression of salt stress induced accumulation of proline.     

Anaerobic stimulation of root exudates and disease of peas

Summary The relationships between root exudation, root disease and anaerobic root stresses were investigated. Sand culture and mist chamber studies demonstrated that low O₂ and high CO₂ reduced plant growth and increased the exudation of ethanol, amino acids, and sugars by pea roots. The relative loss of ethanol by roots was much greater in treatments with atmospheres of N₂ containing 30% CO₂ than in treatments of air containing 30% CO₂ or N₂. Ethanol was not detected in the nutrient solution of aerated plant roots. Atmospheres of N₂ plus 30% CO₂ caused 500% greater mycelial growth ofFusarium solani f. sp.pisi and 400% more disease of inoculated pea roots. Relative losses of four amino acids and four sugars were much greater in atmospheres of N₂ plus 30% CO₂ than in N₂ or air.     

Sugar and Amino Acid Content of Poa pratensis Infected with Ustilago striiformis and Urocystis agropyri

Leaf and root tissues of Poa pratensis L. showing distinct morphological changes associated with infection by Ustilago striiformis (West.) Niessl var. poae (stripe smut) or Urocystis agropyri (Pruess) Schroet. (flag smut) were analyzed for total soluble sugars and free amino acids. Decreases in the quantity of soluble sugars in stripe-smutted plants were not significant, indicating that the presence of U. striiformis sori in leaf tissue does not interfere with photosynthesis of the host. Severe decreases in the quantity of soluble sugars in flag-smutted plants suggest that U. agropyri either directly impairs photosynthesis or effectively metabolizes the photosynthate. Both pathogens caused significant decreases in total free amino acids in leaf and root tissues. The negligible decrease in soluble sugars in stripe-smutted plants was associated with a disproportionate decrease in free amino acids, suggesting that the pathogen either metabolizes amino acids or inhibits their synthesis. The severe decrease in amino acids in leaves and roots of U. agropyri-infected plants is believed due to carbohydrate starvation. It is probable that morphological changes in U. agropyri-infected plants, including reduced branching, dwarfed leaves and root systems, and the inability to produce inflorescences, are probably the direct result of severely reduced levels of sugars and amino acids. The reduced branching, somewhat smaller root systems, and inhibition of inflorescence production on plants infected by U. striiformis probably are associated to some extent with decreases in amino acids. The upright elongated leaves of U. striiformis-infected plants, however, cannot be attributed to the decrease in amino acid content and are suggestive of hyperauxiny.     

Effect of bioaccumulation of cadmium on biomass productivity,essential trace elements,chlorophyll biosynthesis,and macromolecules of wheat seedlings

Soil contamination with heavy metals has become a worldwide problem, leading to losses in agricultural yield and hazardous human health effects as they enter the food chain. The present investigation was undertaken to examine the influence of cadmium (Cd²⁺) on the wheat (Triticum aestivum L.) plant. Cd²⁺ accumulation and distribution in 3-wk-old seedlings grown in nutrient medium containing varying concentrations of Cd²⁺ (control, 0.25, 0.50, 1.0, 2.5, and 5.0 mg/L) was monitored. The effect of varying Cd²⁺ concentrations up to 21 d on biomass productivity, plant growth, photosynthetic pigments, protein, amino acids, starch, soluble sugars, and essential nutrients uptake was studied in detail to explore the level up to which the plant can withstand the stress of heavy metal. Plants treated with 0.5, 1.0, 2.5, and 5.0 mg/L Cd²⁺ showed symptoms of heavy-metal toxicity as observed by various morphological parameters which were recorded with the growth of plants. The root, shoot-leaf length and the root, shoot-leaf biomass progressively decreased with increasing Cd²⁺ concentration in the nutrient medium. Cd²⁺ uptake and accumulation was found to be maximum during the initial growth period. Cd²⁺ also interfered with the nutrients uptake, especially calcium (Ca²⁺), magnesium (Mg²⁺), potassium (K⁺), iron (Fe²⁺), zinc (Zn²⁺), and manganese (Mn²⁺) from the growth medium. Growth reduction and altered levels of major biochemical constituents such as chlorophyll, protein, free amino acids, starch, and soluble sugars that play a major role in plant metabolism were observed in response to varying concentrations of Cd²⁺ in the nutrient medium. In the present study, the effects of Cd²⁺ on growth, biomass productivity, mineral nutrients, chlorophyll biosynthesis, protein, free amino acid, starch, and soluble sugars in wheat plants was estimated to establish an overall picture of the Cd²⁺ toxicity at structural and functional levels.     

External Nitrogen and Carbon Source-Mediated Response on Modulation of Root System Architecture and Nitrate Uptake in Wheat Seedlings

Nitrogen uptake efficiency is an important component trait that could be targeted for improving nitrogen use efficiency of crop plants. To understand the responses of different nitrate transport systems and the influence of root system architecture on nitrate uptake under limited nitrate conditions in wheat (Triticum aestivum L.) at the seedling stage, we studied nitrate uptake, root system architecture, and expression of different nitrate transporter genes in induced and non-induced wheat seedlings. Further, effects of inclusion of sucrose and two amino acids (glutamine and asparagine) in induction medium on these parameters were also studied. We observed that the induced wheat root system took up more nitrate as compared to non-induced root system in a dose-dependent manner. Gene expression of both high- and low-affinity nitrate transporter gene showed differential expression in the induced root tissues, as compared to non-induced tissues, depending on the concentration of nitrate present in induction medium. External nutrient media containing sucrose, glutamine, and asparagine reduce nitrate concentration in both root and shoot tissues and also influence the gene expression of these transporters. Our observations indicate that upon induction with milder external nitrate concentrations, the root architecture is modulated by changing overall lateral root size and 1st order lateral root numbers along with activation of nitrate transporters which acquire and transport nitrate in roots and shoots, respectively, depending on the carbon and nitrogen source available to seedlings.

     

Purification and physicochemical properties of α -amylase from irradiated wheat

α-Amylases from control and gamma-irradiated (at 0.2 and 2.0 kGy dose levels) wheat seedlings were purified to homogeneity and characterized. The molecular weight of the enzyme from a 2 kGy irradiated sample was slightly lower than that of the control; other general and catalytic properties also showed some differences. α-Amylase from the irradiated (2 kGy) sample had a narrow range of pH optimum and was inactivated faster at alkaline pH and by heat treatment than the enzyme from unirradiated wheat. A high apparent Michaelis constant (K _m) and a low maximal velocity (V_max) for the hydrolysis of soluble starch catalyzed by the enzyme from irradiated (2 kGy) wheat, suggested some modifications in the formation of the substrate α-amylase complex. Further, of the total number of amino acid residues lost on irradiation, dicarboxylic amino acids constituted the largest percentage; these structural alterations in the enzyme may be responsible for its partial inactivation. The total sugars liberated upon amylolysis of starch with the 2 kGy irradiated enzyme were lower than control, and there was accumulation of higher maltodextrins in the place of maltose.     

Effects of Sulphur Dioxide on Sugar and Free Amino Acid Content of Pine Seedlings

Treatment of jack pine (Pinus banksiana Lamb.) seedlings with gaseous SO₂ resulted in a shift between the reducing and non-reducing sugars. Increasing concentrations of gaseous SO₂ caused an increase in reducing sugars and a decline in the non-reducing sugars, suggesting a conversion from the latter to the former at high SO₂ concentrations. The total amino acid content of the intact tissues also increased with increasing concentrations of gaseous SO₂. Gas-liquid chromatographic analyses of the amino acids indicated that SO₂ (1. 34 mg · m^-3 for 96 h) resulted in an increase in the content of alanine, valine, glycine, isoleucine, leucine, threonine, aspartic acid tyrosine, lysine, and arginine, and a decrease in the content of serine and glutamic acid. The enzymatic and other implications of such changes are discussed.     

Root exudates of plants

Summary Comparisons have been made of the amount and composition of seed and seedling exudates in barley, wheat, cucumber, and bean. Except in the case of wheat a greater proportion of the total nitrogen content in the seed exudates was formed by protein and peptide nitrogen than by nitrogen of free amino acids. In contrast, the greater part of the total nitrogen in the exudates of seedlings was formed by free amino acid nitrogen, except in the case of barley. Peptides represented 8 to 26 per cent of the protein and peptide fraction in seed exudates, the highest amount being found in bean. On an equal weight basis, the spectrum of amino acids released from seeds and seedlings differed little between barley, wheat and cucumber; greater differences were observed in bean exudates. Seedlings exuded reducing substances to a considerably greater extent than seeds. The spectrum of reducing sugars in seed and root exudates differed greatly, especially in the case of keto sugars. Differences in the organic acid spectra were small, except for bean plants, the seedling exudate of which contained more organic acids with a richer spectrum than seed exudate. Both seeds and seedlings of cucumber exuded a small quantity and a poor spectrum of organic acids.     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

The importance of root carbohydrate abundance in ammonium uptake

The influence of carbohydrates on ammonium uptake and ammonium transporter (AMT1) expression was investigated in roots of field pea (Pisum arvense) and rutabaga (Brassica napus var. rapifera). Ammonium transport into field pea seedlings diminished markedly following cotyledon removal, which indicated that uptake of ammonium was under control of reserves stored in the cotyledons. Excision of cotyledons decreased also the level of some amino acids, glucose and total reducing sugars in field pea roots. To investigate the importance of the sugar supply for the regulation of ammonium uptake at low external NH ₄ ⁺ level, 1 mM glucose or sucrose was supplied for several hours to the field pea seedlings deprived cotyledons or to intact rutabaga plants. Supply of both sugars resulted in a substantial increase in ammonium uptake by both plant species and enhanced markedly the expression of AMT1 in rutabaga roots. The results indicate that sugars may regulate ammonium transport at the genetic level.     

Chemotaxis of Rhizobium SP.S2 Towards Cajanus cajan Root Exudate and Its Major Components

The chemotactic response of Rhizobium sp. S2, a slow-growing Cajanus cajan isolate, towards its host root exudate was examined. Two classes of mutants, one nonchemotactic towards nutrients (amino acids and sugars) and signal compounds like flavonoids and the other, nonchemotactic towards amino acids and sugars but positive towards naringenin, the flavonoid present in Cajanus cajan root exudate, were obtained. The plasmid-cured derivative of the parent showed positive response towards amino acids and sugars but was nonchemotactic towards naringenin. A possible presence of dual chemotaxis pathways, one towards nutrients and the other for sensing signal compounds, was thus demonstrated. The possible involvement of naringenin as a chemoattractant in the preliminary stages of this Rhizobium-legume interaction was also established. Received: 18 September 1998 / Accepted: 22 October 1998     

Amino acid uptake by Ricinus communis roots: characterization and physiological significance

Abstract Roots of sterile-grown, intact 6-day-old seedlings of Ricinus communis possess at least two independent active amino acid uptake systems, one for neutral and one for basic amino acids. The kinetics of uptake of L-proline and L-arginine, which were taken as representative substrates for the two systems, are biphasic. At low concentrations (0.01–0.5 mol m^?3) Michaelis -Menten kinetics prevail, changing to a linear concentration dependence at higher substrate concentrations (1–50 mol m^?3). L-glutamate uptake velocity is linear over the whole substrate concentration range. For comparison the uptake kinetics of nitrate and ammonium were determined as well as interactions among the different nitrogen sources. The K_m value for nitrate uptake was 0.4 mol m^?3, and for ammonium 0.1 mol m^?3. The uptake capacity for nitrate or ammonium was approximately the same as for amino acids. The interaction between the uptake systems for organic and inorganic nitrogen is small. Two hypotheses for the physiological significance of amino acid uptake by roots were considered: (i) Uptake of amino acids from the soil-determination of amino acids in soil and in soil water indicates that they might contribute 15–25% to the nitrogen nutrition of the plant. (ii) Amino acid uptake systems of root cells serve primarily as retrieval of amino acids delivered from the phloem- it was found that ¹⁴C L-glutamine, which was delivered to the cotyledon and transported to the root via the phloem, was not lost by the roots, whereas it appeared in the bathing medium if L-glutamine was applied externally to the root to compete for the uptake sites; this suggests that an apoplastic pool of amino acids in the root exists due to their efflux from the phloem.     

Influence of silicon on spring wheat seedlings under salt stress

The aim of the study was to examine the effect of silicon on spring wheat subjected to salt stress. The experiment was conducted in hydroponic conditions on 10-day old wheat seedlings. Salt stress was induced by sodium chloride at the concentration of 70 and 100 mM added to nutrient medium. Silicon (H₄SiO₄) at the doses of 1.0 and 1.5 mM significantly increased the shoots and roots weight of wheat seedlings and the content of photosynthetic pigments (chlorophyll a and b, as well as carotenoids) in leaves. It reduced a detrimental effect of salt stress and restricted peroxidation of membrane lipids. We also observed a greater accumulation of nitrates and the decrease in malondialdehyde concentration in plant tissues as a result of silicon addition. Under osmotic stress, silicon did not change the content of sugars in wheat shoots and roots. Silicon did not clearly affect proline content. In general, the obtained results point out that silicon can be used for the alleviation of adverse effect of salinity on plants status.     

Effects of increasing inorganic carbon supply to roots on net nitrate uptake and assimilation in tomato seedlings

We investigated the influence of an increased inorganic carbon supply in the root medium on NO^?₃ uptake and assimilation in seedlings of Lycopersicon esculentum (L.) Mill. cv. F144. The seedlings were pre-grown for 4 to 7 days with 0 or 100 mM NaCl in hydroponic culture using 0.2 mM NO^?₃ (group A) or 0.2 mM NH⁺₄ (group B) as nitrogen source. The nutrient solution for group A plants was aerated with air or with air containing 4 800 μumol mol^?1 CO₂. Nitrate uptake rate and root and leaf malate contents in these plants were determined. The plants of group B were subdivided into two sets. Plants of one set were transferred either to N-free solution containing 0 or 5 mM NaHCO₃, or to a medium containing 2 mM NO^?₃ and 5 mM NaHCO₃. Both sets of group B plants were grown for 12 h in darkness prior to 2 h of illumination, and were assayed for malate content and NO^?₃ uptake rate (only for plants grown in N-free solution). The second set of group B plants was labeled with ¹⁴C by a 1-h pulse of H¹⁴CO^?₃ which was added to a 5 mM NaHCO₃ solution containing 0 or 100 mM NaCl and 0 or 2 mM NO^?₃, and ¹⁴C-assimilates were extracted and fractionated. The roots of group B plants growing in carbonated medium accumulated twice as much malate as did control plants. This malate was accumulated only when NO^?₃ was absent from the root medium. Both a high level of root malate and aeration with CO₂-enriched air stimulated NO^?₃ uptake. Analysis of ¹⁴C-assimilates indicated that with no NO^?₃ in the medium, the ¹⁴C was present mainly in organic acids, whereas with NO^?₃, a large proportion of ¹⁴C was incorporated into amino acids. Transport of root-incorporated ¹⁴C to the shoot was enhanced by NO^?₃, while the amino acid fraction was the major ¹⁴C-assimilates in the shoot. It is concluded that inorganic carbon fixed through phosphoenolpyruvate carboxylase (EC 4.1.1.31) in roots of tomato plants may have two fates: (a) as a carbon skeleton for amino acid synthesis; and (b) to accumulate, mainly as malate, in the roots, in the absence of a demand for the carbon skeleton. Inorganic carbon fixation in the root provides carbon skeletons for the assimilation of the NH⁺₄ resulting from NO₃ reduction, and the subsequent removal of amino acids through the xylem. This ‘removal’ of NO^?₃ from the cytoplasm of the root cells may in turn increase NO^?₃ uptake.     

Relationships between cell membrane stability, exudate content and infectivity of Bradyrhizobium japonicum strain 639 to boron starved soybean plants

The influence of boron starvation on the root exudates content in soybean seedlings (Glycine max. L. Merr.) and the effect of exudates pretreatment on the pre-infection processes in symbiotic system Br. japonicum strain 636 and soybean were investigated. Root cell membrane stability of boron starved soybean plants (-B) decreased compared to the control. The concentrations of all analyzed metabolites (reducing sugars, free amino acids, organic acids, soluble phenols and total flavonoids) from root exudates of -B plants were lower than the control concentrations. Analysis of polyphenols after HPLC chromatography of root exudates showed significant difference of peak numbers between chromatograms of exudates obtained from boron starved and from control plants. Bacterial culture treatment with root exudates from -B plants showed decreased growth, chemotaxis and attachment ability toward the host root compared to the control exudate treatments. These changes were accompanied by decreased nodulation and acetylene reduction activity of boron starved soybean plants.     

Physiological characterization and electron microscopic investigation of cyanobacteria associated with wheat rhizosphere

Physiological attributes of a set of cyanobacterial strains, isolated from the rhizosphere of wheat (var. HD 2687), identified as belonging to the genera Calothrix (n = 3), Westiellopsis (1), Hapalosiphon (2) and Nostoc (2), were axenized and evaluated. The concentrated culture filtrates of three cyanobacterial strains — C. ghosei, H. intricatus and Nostoc sp. were able to enhance germination percentage, radicle and coleoptile length in inhibition experiments with wheat seeds. Indole-3-acetic acid (IAA) production was recorded in light and dark (+0.5 % glucose) incubated cultures. Incubation in the presence of tryptophan significantly enhanced IAA production. Acetylene-reducing activity was higher in light incubated cultures of Nostoc sp. followed by C. ghosei, while in the dark, C. ghosei recorded highest values. TLC of the filtrates revealed the presence of several amino acids such as histidine, and auxin-like compounds. Co-culturing with selected strains recorded significant enhancement in plant chlorophyll. Root sections of wheat seedlings co-cultured with C. ghosei revealed the presence of short filaments inside the root hairs and cortical region. Such strains can be promising candidates for developing plant growth promoting associations for wheat crop, besides serving as model systems for understanding the metabolic interactions of cyanobacteria with host plant, such as wheat.     

Chemotaxis ofRhizobium sp. Towards root exudate ofCicer Arietinum L.

Summary Root exudate from seedlings ofCicer arietinum L. was collected in a chamber under aseptic conditions. The exudate was fractionated into anion, cation and neutral fractions. The anionic fraction was made up of galacturonic acid, gluconic acid, mannuronic acid and two unidentified compounds withR _f values 0.56 and 0.62. The cationic fraction contained alanine, arginine, aspartic acid, cystine, glycine, histidine, isoleucine, leucine, lysine and serine. The neutral fraction was made up of arabinose, galactose, glucose, ribose and xylose. The amino acids contributed to the bulk of the root exudate. The ratio of anionic, cationic and neutral fraction was 1∶7∶2. The crude root exudate was tested for its chemotactic ability using the capillary tube method. It was highly chemotactic for theRhizobium sp. The individual fractions and their various combinations were tested for chemotaxis. The chemotactic response of the Cicer strain of Rhizobium was least with anionic fraction most with cationic fraction and intermediate with neutral fraction. Maximum chemotactic response among the fractional combinations was obtained with all the three fractions and least with cationic plus neutral factions. Individual compounds constituting the various fractions were also tried for their ability to elicit chemotactic response. The organism exhibited maximum positive chemotactic response to histidine and negative response to alanine among the amino acids and to glucose and gluconic acid among the sugars and sugar acids.     

Pectolytic enzymes activity and pathogenicity ofGaeumannomyces graminis var.tritici and related Phialophora-like fungi

Gaeumannmyces graminis var.tritici (Ggt), Phialophora sp. (lobed hyphopodia) andPhialophora graminicola vere grown in a liquid medium with pectin and on autoclaved wheat roots (root media) and the activity of pectolytic enzymes in culture filtrates was measured. Most strains of the fungi exhibited polygalacturonate trans-eliminase activity but no pectin methylesterase activity was detected.Ggt polygalacturonase was found in culture filtrates from all the media used whilePhialophora sp. did not exhibit activity of this enzyme in the unbuffered root media. No polygalacturonase activity was demonstrated forP. graminicola. A correlation was found (r=0.548) betweenin vitro polygalacturonase activity and the pathogenicity ofGgt to wheat seedlings.     

Influence of Nitrogen and Sucrose in the Medium and of Irradiance of the Stock Plants on Root Formation in Pelargonium Petioles Grown in vitro

In vitro root formation was studied in petiole explants from Pelargonium×hortorom Bailey cv. Radio exposed to different irradiance (2.5. 11.6 or 23.0 W/m²). Optimal root formation was found when stock plants were subjected to 2.5 W/m² and explants were grown on a medium with 20 g/I of sucrose and 5.0 mM of nitrogen. The number of roots per explant decreased with increasing nitrogen or sucrose concentrations in the growth medium and with increasing irradiance applied to the stock plants. Variation in temperature (17, 21 or 25°C) did not affect root formation. The number of roots per explant was higher in explants from young petioles than in explants from older petioles. The initial nitrogen content of the explants was highest at the lowest irradiance. The content of reducing sugars and sucrose increased with increasing irradiance. The endogenous levels of lysine, arginine and ornithine were highest at 2.5 W/m² and the levels of proline. alanine, γ-aminobutyric acid, glutamic acid, glutaminc, aspartic acid and asparagine were highest at 23.0 W/m². With rising nitrogen concentration in the medium, the final endogenous content of nitrogen in the explants increased, whereas the content of reducing sugars decreased. When the sucrose concentration in the medium increased, the endogenous content of reducing sugars rose, and the nitrogen level was lowered. Variations in the nitrogen concentration in the growth medium influenced the final level of free amino acids in the explants.