SSSAJ: Volume 71: Number 1 • January –February 2007 75

Soil Sci. Soc. Am. J. 71:75-85doi:10.2136/sssaj2006.0055Received 9 Feb. 2006.*Corresponding author (amuscolo@unirc.it).© Soil Science Society of America677 S. Segoe Rd. Madison WI 53711 USA

Humic substances (HS), the largest constituent of soil organic matter (?60%), are a key component of the ter-

restrial ecosystem, being responsible for many complex chemi-cal reactions in soil (Stevenson, 1994).

Humic substances are known to increase soil aggregation (Bremner, 1954; Whitehead, 1963), compactability (Soane, 1990), water-holding capacity (De Jong et al., 1983) and the cat-ion exchange capacity (Stevenson, 1994). Directly, HS promote plant growth by acting on membrane permeability as protein carriers of ions, activating respiration, the Krebs cycle, photo-synthesis, and the production of adenosine triphosphate and amino acids (Malcolm and Vaughan, 1978, 1979; Vaughan and Malcolm, 1985). Humic matter has a very complex biological activity, depending on its origin, molecular size, chemical charac-teristics, and concentration. It exhibits a range of different effects

on plant metabolism in the diverse systems that have been tested (Maggioni et al., 1987; Albuzio et al., 1993; Nardi et al., 2002). During the last 10 yr, we have investigated the biological activity of HS derived from different soils (Dell’Agnola and Nardi, 1987; Muscolo et al., 1993; Nardi et al., 1994, 1998, 1999; Concheri et al., 1996; Muscolo et al., 1996, 1998, 1999a, 1999b, 2001a, 2001b; Muscolo and Nardi, 1997; Muscolo and Sidari, 1998; Panuccio et al., 2001), showing that HS induce morphogenetic and biological changes in leaf explants of Nicotiana plumbagini-folia, affecting the patterns of peroxidase and esterase, enzymes that are involved in organogenesis and may be indicators of somatic embryogenesis. These effects, peculiar to the humic frac-tion with a low relative molecular mass (<3500 Da), were simi-lar to those produced by indole-3-acetic acid (IAA); the humic fraction with a high relative molecular mass (>3500 Da) had no effect on this vegetable system. A study on homogeneous carrot (Daucus carota L.) cell cultures compared the effects of the low-relative-molecular-mass humic fraction to different auxins. This humic fraction caused an increase in carrot cell growth similar to that induced by 2,4-dichlorophenoxyacetic acid (2,4-D) and promoted morphological changes similar to those induced by IAA. It is clear from this that low-molecular-weight components

Biological Activity of Humic SubstancesIs Related to Their Chemical Structure

To understand if the biological activity of humic substances may be related to their molecular weight or chemical structure, two humic substances, derived from an Ah horizon of unculti-vated couch grass [Elytrigia repens (L.) Desv. ex Nevski] and an Ah horizon of forest soil, were extensively characterized by means of different spectroscopic techniques (diffuse refl ectance infrared Fourier transform [DRIFT] and 1H nuclear magnetic resonance [NMR]). The two humic substances, each separated in fractions with low (<3500 Da) and high (>3500 Da) relative molecular mass were compared for their effects on Pinus nigra J.F. Arnold callus. Growth of callus, the soluble sugar content, free amino acid pool, and the activities of the key enzymes involved in C and N metabolism were investigated. Callus was grown for a subculture period (28 d) on basal Murashige and Skoog medium plus humic matters with or without different hormones: indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, or 6-ben-zylaminopurine. The results of 1H-NMR spectra and the DRIFT spectroscopy showed sig-nifi cant differences in the chemical composition between forest and grass humic substances. A large amount of aliphatic and H-sugarlike component and an intense chemical shift of the β-CH3 region in both grass humic fractions were observed, while high contents of betaine, organic acid, and COOH groups in both forest humic fractions were detected. A different biological activity between the grass and forest humic fractions was also observed. Thus, the different activity of the two humic substances used seems related to the diverse chemical composition rather than to different molecular weights.

Adele Muscolo*Maria SidariEmilio AttinàDipartimento di Gestione dei Sistemi Agrari

e ForestaliUniversità degli Studi MediterraneaFeo di Vito89060 Reggio CalabriaItaly

Ornella FranciosoDipartimento di Scienze e Tecnologie

AgroambientaliUniversità di BolognaVia Fanin 4040127 BolognaItaly

Vitaliano TugnoliDipartimento di BiochimicaUniversità di BolognaVia Belmeloro 8/240126 BolognaItaly

Serenella NardiDipartimento di Biotecnologie AgrarieFacoltà di Agraria, AgripolisStrada Romea 16–35020Legnaro, PadovaItaly

SOIL C

HEM

ISTRY

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; ALD, aldolase; BAP, 6-benzylaminopurine; DRIFT, diffuse refl ectance infrared Fourier transform; GDH, NAD(H) glutamate dehydrogenase; GK, glucokinase; GOGAT, glutamate synthase; HMWF, high-molecular-weight humic substance derived from forest soil; HMWG, high-molecular-weight humic substance derived from couch grass soil; HS, humic substances; IAA, indole-3-acetic acid; LMWF, low-molecular-weight humic substance derived from forest soil; LMWG, low-molecular-weight humic substance derived from couch grass soil; MDH, malate dehydrogenase; NAD+, nicotinamide adenine dinucleotide; NMR, nuclear magnetic resonance; PEPC, phosphoenolpyruvate carboxylase; PGI, phosphoglucoisomerase; PK, pyruvate kinase; SAI, soluble acid invertase.

76 SSSAJ: Volume 71: Number 1 • January –February 2007

of HS are biologically active (Vaughan, 1967; Mato et al., 1972; Vaughan et al., 1974; Piccolo et al., 1992; Muscolo et al., 1998; Nardi et al., 2000), even if, in some experimental models, high-molecular-weight components appeared to be similarly active (Ladd and Butler, 1971; Malcolm and Vaughan, 1979).

The mechanism by which soil HS stimulate plant biologi-cal activity is not well clarifi ed; this is in part due to their het-erogeneity and to the diffi culty of their characterization. Thus attempts to relate humus structure to biological activity have produced contrasting results. Although the molecular structure has not yet been extensively defi ned, according to the new view, HS are collections of diverse, relatively low molecular mass com-ponents forming dynamic associations stabilized by hydropho-bic interactions and H bonds (Piccolo et al., 1996; Conte et al., 2003; Spaccini et al., 2006). These associations are capable of organizing, in suitable aqueous environments, into supramolec-ular structures stabilized by dispersive interactions and H bond-ing. Functional carboxylic and phenolic C groups in HS seem to have an important role in determining their biological activ-ity (Mato et al., 1972; Malcolm and Vaughan, 1978; Pfl ug and Ziechmann, 1981), but the manner in which they act has still to be elucidated (Vaughan and Malcolm, 1985). Although it is very diffi cult to defi ne a clear concept of their composition (Hayes, 1997), considerable progress has been made in the last few years in providing an awareness of some of the gross features of HS by using various spectroscopic procedures: infrared spectroscopy, electron spin resonance, Raman, ultraviolet-visible, fl uorescence, and x-ray photoelectron spectroscopy. In recent years, consid-erable effort has been focused on 13C-NMR and 1H-NMR spectroscopy, considered the most powerful tools available to determine the structure of both organic and inorganic species.

To clarify if the biological activity of HS may be related to their chemical composition or molecular weight, 1H-NMR and DRIFT spectroscopy were used to obtain detailed chemical information that could be of use to elucidate the mechanism of action of HS. For this purpose, callus, a coherent and amorphous tissue, was used to study in vitro the effects of HS without environmental interference.

The use of tissue culture provides a rapid and inexpensive method to screen compounds and has been found to be repre-sentative of results obtained in whole-plant experiments study-ing different aspects (Souissi and Kremer, 1998; Ehsanpour and Fatahian, 2003; Vidal et al., 2004).

We compared the biological effects of two HS derived from an uncultivated couch grass fi eld and a forest soil, both separated into high (>3500 Da) and low (<3500 Da) relative molecular mass fractions, on Pinus nigra callus growth. Biochemical parameters, such as sucrose, glucose, and fructose content, free amino acid pool, and the activity of the key enzymes of C and N metabolism were investigated.

MATERIALS AND METHODSStudy Sites

The study was performed on the Ah horizon of a Typic Eutrudept (Soil Survey Staff, 1992) of uncultivated couch grass fi elds located on the College of Agriculture Farm (University of Padua), and on the Ah horizon of a Typic Hapludalf (Soil Survey Staff, 1992) from a site near Pian Cansiglio (Tambre, Belluno, Italy) characterized by a Fagus sylvatica L. subsp. sylvatica vegetal cover.

Humic Substances ExtractionHumic substances were extracted from the air-dried samples with

0.1 M KOH (1:20 w/v) at room temperature for 16 h under an N2 atmosphere and were freed from the suspended material by centrifu-gation at 7000 × g for 20 min. Humic extracts were transferred into 18 000 molecular weight cutoff dialysis tubings Visking (Medicell, London) and dialyzed against double-distilled water. The retained solution was desalted by ion exchange on Amberlite IR 120 (H+ form, Aldrich Chemical Co., Milwaukee, WI) and this fraction was treated with glacial acetic acid until pH 2.1 was attained. The acidifi ed solution was dialyzed through 3500 molecular weight cutoff Spectra/Por 3 tub-ing (Spectrum, Gardena, CA) against distilled water. This procedure sepa-rated the total humic extract into high (>3500 Da) and low (<3500 Da) relative molecular mass (Mr) fractions. The high-molecular-weight humic fractions and low-molecular-weight humic fractions were collected inside and outside the dialysis tubing, respectively. These solutions were freed from unreacted acetic acid and reduced in volume to about 50 mL by vacuum distillation (Nardi et al., 1991).

Fourier-Transform Infrared SpectroscopyFor each analysis, 2 mg of dried sample was mixed with 148 mg

of KBr (Fourier-transform infrared grade, Aldrich Chemical Co.), so that the mixture became homogeneous. After grinding, the sample mixture was heaped over the top of the micro sample cup. Any excess material was removed with a straight-edged tool. For the background, a micro sample cup of pure KBr was prepared. The spectra were recorded with a Nicolet Impact 400 Fourier-transform infrared spec-trophotometer (Nicolet Instruments, Madison, WI) and fi tted with an apparatus for diffuse refl ectance (Spectra-Tech, Stamford, CT). Spectra were recorded with 200 scans collected at 4 cm−1 resolution. Spectra were collected and manipulated by using the Omnic (3.1) software supplied by Nicolet Instruments.

Hydrogen-1 Nuclear Magnetic Resonance Spectroscopy

Humic fractions (20 mg) were dissolved in 0.5 mL D2O (deuterium oxide). The spectra were recorded with a Bruker ACF 250 spectrometer (Bruker Optics, Bellerica, MA) using a 5-mm multinuclear probe. The 1H spectra were accumulated with 16-K data point, one pulse sequence, 40° pulse angle, 3-s relaxation delay, and a sweep width of 2.5 kHz. To obtain a satisfactory signal to noise ratio, 1000 to 2000 scans were needed. Gated irradiation was applied between acquisitions to presaturate the residual water peak. A 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt, was added to the samples to provide a chemical shift standard (Francioso et al., 2000). The spectra were divided into three main regions: aromatic H from 6.0 to 8.0 ppm; H attached to O groups in C α from 4.2 to 3.0 ppm (also defi ned as sugarlike), and aliphatic H from 3.0 to 0.5 ppm. In accordance with results obtained by Wilson et al. (1983), the sugar-like region was attributed to protons largely arising from polysaccharides. Wilson et al. (1983) and Simpson et al. (1997) showed that the aliphatic region might be affected by the presence of protons attached to aromatic rings in α, β, and γ positions.

Callus GrowthExcised leaves of P. nigra were grown in petri dishes, on MS

medium (Murashige and Skoog, 1962) used as basal medium, sup-plemented with 3% sucrose, 0.5 mg L−1 2,4-D, and 0.25 mg L−1 6-benzylaminopurine (BAP) for 35 d to induce callusing. The pH was adjusted to 5.7 before adding 0.8% w/v Bacto agar (Oxoid 1), and the

SSSAJ: Volume 71: Number 1 • January –February 2007 77

media were autoclaved for 20 min at 121°C. After 35 d, callus tissue was transferred for 28 d to the basal medium culture free (control) or with two humic fractions added that were derived from uncultivated couch grass—LMWG with low Mr (<3500 Da) or HMWG with high Mr (>3500 Da), or two forest humic fractions—LMWF with Mr < 3500 Da or HMWF with Mr > 3500 Da, or BAP (0.25 mg L−1), or IAA (2.0 mg L−1), or 2,4-D (0.5 mg L−1), or 2,4-D + BAP (0.5+0.25 mg L−1). Both fi lter-sterilized forest and grass humic fractions were used at a con-centration of 1 mg C L−1, according to previous tests showing that, in the range of 0.1 to 5.0 mg C L−1, 1.0 mg C L−1 was the concentration of humic matter that most interfered with P. nigra callus (Muscolo et al. 2005). In addition, 1 mg C L−1 is the concentration of HS com-monly present in forest soils (Cheng et al., 1996). All treatments were performed at 25°C in darkness. Samples of callus were taken at the end of the period (28 d) for all treatments for enzymatic analysis. The data are the means of fi ve replicates.

Growth ParametersTwenty-eight days after the beginning of the treatment, the callus

growth was estimated as fresh weight. After weighing, the mean relative growth rate (RGR) was calculated as: RGR = ln (FW2 − FW1)/t2 − t1, where FW1 is the fresh weight at the beginning of the measurements, FW2 is the fresh weight at the end of the experiments (28 d), t1 is time at the beginning of the experiments, and t2 is time at the end of the experiments (Hunt, 1990).

Soluble Sugars DeterminationCallus tissue was extracted three times with boiling 80% ethanol

(v/v). Homogenates were centrifuged at 12000 × g and the ethano-lic extract was evaporated under vacuum (Rotavapor RE 111, Büchi, Switzerland), resolubilized in distilled water, and subjected to enzy-matic assay. Glucose, fructose, and sucrose were determined by using hexokinase, glucose-6-phosphate dehydrogenase, and phosphoglucose isomerase, respectively, and after enzymatic inversion to d-glucose and d-fructose by the enzyme β-fructosidase (Boehring test combination 716 260; Bergmeyer and Bernt, 1974). Absorbance was detected at 340 nm (UV-2100 spectrophotometer, Shimadzu, Japan).

Measurement of Free Amino AcidsThe total free amino acid pool was determined by ninhydrin assay

(Yemm and Cocking, 1955). Absorbance readings were converted to grams amino acid per kilogram fresh weight using a glycine standard curve.

Enzyme Extraction and Assay ConditionsCallus tissue was extracted according to the method of Zhifang et al.

(1999), modifi ed as follows: samples of callus tissues (?1 g fresh weight) were homogenized in a chilled mortar with three volumes of chilled extrac-tion buffer containing 100 mM HEPES-NaOH (pH 7.5), 5 mM MgCl2, and 1 mM dithiothreitol. The extract was fi ltered through two layers of muslin and clarifi ed by centrifugation at 20000 × g for 15 min. The super-natant was used for enzymatic analysis. All steps were performed at 4°C.

Soluble acid invertase (SAI, β-fructofuranosidase, β-fructofu-ranoside fructohydrolase, EC 3.2.1.26) activity was assayed at 37°C by adding 50 μL of extract to 50 μL of 1 M NaOAc (pH 4.5). The enzyme reaction was started by the addition of 100 μL of a 120 mM sucrose solution. The reaction was stopped at 30 min by adding 30 μL of 2.5 M 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS, Trizma base) and boiling the mixture for 3 min. The concentration of glucose

liberated was determined with a glucose test kit from Sigma-Aldrich (St. Louis, MO; Zhu et al., 1997).

Glucokinase (GK: d-glucose-6-phosphotransferase, EC 2.7.1.1) activity was measured by coupling hexose phosphate production with nicotinamide adenine dinucleotide (NAD+) reduction by glucose-6-phosphate dehydrogenase and monitoring at absorbance A340 (Huber and Akazawa, 1986).

Phosphoglucoisomerase (PGI, d-glucose-6-phosphate ketoisomer-ase, EC 5.3.1.9) assay contained 50 mM Hepes–NaOH (pH 7.2), 5 mM MgCl2, 5 mM fructose-6-phosphate, 1 mM NAD+, and 1 IU mL−1 glu-cose-6-phosphate dehydrogenase. The activity was measured spectrophoto-metrically at A340 (Tsai et al., 1970).

Aldolase (ALD, EC 4.1.2.13) was assayed spectrophotometrically at A340. The assay contained 50 mM Hepes–NaOH (pH 7.2), 5 mM MgCl2, 1 mM fructose-1,6-bis phosphate, 0.2 mM NADH, 1 IU mL−1 glycerol-3-phospho-dehydrogenase, 1 IU mL−1 triose phosphate isomer-ase, and 50 μL extract (Doehlert et al., 1988).

Pyruvate kinase (PK, EC 2.7.1.40) was assayed by adding to 450 μL 0.1 M triethanolamine (TEA), adjusted with 0.1 M NaOH to pH 7.75, 50 μL of 3 mM β-NADH-Na2 in 0.1 M TEA (pH 7.75), 50 μL of 52 mM adenosine 5′-diphosphate-Na2 in 0.1 M TEA (pH 7.75), 50 μL of 0.15 M MgSO4⋅6H2O, 50 μL of 0.15 M KCl in 0.1 M TEA (pH 7.75), 50 μL of l-lactic dehydrogenase, and 50 μL of extract. The reaction was started after a lag time of 10 min at 30°C by adding 50 μL of 0.225 M 2-phosphoenol-pyruvate–Na–H2O in 0.1 M TEA (pH 7.75) (Bergmeyer et al., 1986).

Glutamate synthase (GOGAT, EC 1.4.7.1) assay contained 25 mM HEPES-NaOH (pH 7.5), 2 mM L- glutamine, 1 mM α-ketoglutaric acid, 0.1 mM NADH, 1 mM Na 2 EDTA, and 100 μL of enzyme extract. GOGAT was assayed spectrophotometrically by monitoring NADH oxidation at A340 (Avila et al., 1987).

To assay glutamine synthetase (GS, EC 6.3.1.2), the mixture for the transferase assay contained 90 mM imidazole-HCl (pH 7.0), 60 mM hydroxylamine (neutralized), 20 mM Na2HAsO4, 3 mM MnCl2, 0.4 mM adenosine diphosphate (ADP), 120 mM glutamine, and the appro-priate amount of enzyme extract. The assay was performed in a fi nal vol-ume of 750 μL. The enzymatic reaction was developed for 15 min at 37°C. The γ-glutamyl hydroxamate was colorimetrically determined by addition of 250 μL of a mixture (1:1:1) of 10% (w/v) FeCl3⋅6H2O in 0.2 M HCl, 24% (w/v) trichloroacetic acid, and 50% (w/v) HCl. The optical density was recorded at A540 (Canovas et al., 1991).

Malate dehydrogenase (MDH, EC 1.1.1.37) was assayed at 25°C. The assay contained, in 3.17 mL, 94.6 mM phosphate buffer (pH 6.7), 0.2 mM NADH, 0.5 mM oxalacetic acid, and 1.67 mM MgCl2. The MDH was assayed spectrophotometrically by monitor-ing NADH oxidation at A340 (Bergmeyer et al., 1986).

The activity of NAD(H) glutamate dehydrogenase (GDH, EC 1.4.1.3) was measured at 340 nm at 30°C. The assay contained 150 mM tris-HCl at pH 8.0, 100 mM NH4Cl, 10 mM α-ketoglutaric acid–KOH at pH 7.4, 0.3 mM NADH, and 100 μL of enzyme extract in a fi nal vol-ume adjusted to 1 mL with H2O (Refouvelet and Daguin, 1999).

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) enzy-matic activity was spectrophotometrically measured by monitoring NADH oxidation at 340 nm for 5 min at 30°C. The assay medium (1 mL) contained 100 mM tris-HCl pH 8.0, 10 mM MgCl2, 10 mM NaHCO3, 0.2 mM NADH, 1.5 IU MDH, and 100 μL of enzyme extract (Pasqualini et al., 2001).

78 SSSAJ: Volume 71: Number 1 • January –February 2007

Statistical AnalysisThe reported data represent mean values of fi ve replicates.

Comparisons between the means were made using the Student–Newman–Keuls test (Sokal and Rohlf, 1969).

RESULTSHydrogen-1 Nuclear Magnetic Resonance Spectroscopy

The spectra of humic fractions from grass are shown in Fig. 1. The spectra were characterized by two different spec-troscopic patterns. The HMWG showed an aromatic pro-ton region poorly resolved and an intense and broad region attributed to sugarlike and polyether components (Wilson et al., 1983; Wershaw, 1985). Inspecting the aliphatic region, an intense doublet at 1.33 ppm that was identifi ed as a proton in

β-CH3 of lactate appeared (Wilson et al., 1988, Fan, 1996; Francioso et al., 2000). Moreover, other broad and intense resonances at 0.83 and 1.3 ppm are due to the protons of terminal methyl groups of methylene chains (Malcolm, 1990). In particular, two intense resonances appeared at 2.9 and 3.40 ppm and were assigned to protons in α-CH3 of acetoacetate and ether aliphatics, respectively (Pouchert and Behnke, 1993; Fan, 1996). On the contrary, the spectrum of LMWG was characterized only by a broad and unre-solved region assigned to sugarlike components; how-ever, in the spectrum we can see some resonances due to the presence of low-molecular-weight organic sub-stances such as lactate (1.33 ppm) and acetoacetate (2.9 ppm) and a singlet at 1.47 ppm assigned to a proton in β-CH3 of alanine. The aromatic region did not show resonances due to aromatic protons.

In the spectra of the humic fractions from the forest soil, the chemical shifts between 4.0 and 2.0 ppm were attributed to sugarlike and aliphatic groups linked to elec-tronegative atoms (O or N; Fig. 2). Moreover, an intense

resonance in the region 1.8 to 0.6 ppm (Fig. 3) was assigned to protons of –CH3 groups and terminal methyl groups of methylene chains, respectively (Malcolm, 1990).

The HMWF and LMWF spectra (Fig. 2) were character-ized by a different content in sugarlike and low-molecular-weight organic substances. In particular, in the HMWF spectrum there appeared a broad sugarlike band and two singlets at 3.87 ppm, assigned to a proton in α-CH2, and at 3.24 ppm due to a proton in N-CH3 of glycinebetaine (Fan, 1996). Two quar-tets between 2.6 and 2.8 ppm were assigned to different forms of citrate (Fan, 1996). In the LMWF spectrum, the sugarlike region appeared less intense and characterized by different sim-ple sugars. Glycinebetaine was also present, but with low relative intensity with respect to the HMWF fraction. Moreover, in this spectrum there appeared well-resolved signals corresponding to a

singlet at 3.55 ppm of glycine, a singlet at 3.44 ppm of trimethyamine-N-oxide (Fan, 1996), a singlet at 2.39 ppm of succinate, a singlet at 2.37 of pyruvate, and fi nally, a singlet at 2.1 ppm of acetate (Fig. 2). The aliphatic region of HMWF (Fig. 3) was characterized by intense resonance of lactate (1.33 ppm), while the aliphatic protons were not well resolved. The LMWF spectrum (Fig. 3) showed a doublet at 1.47 ppm, assigned to alanine, and an intense resonance around 1.33 ppm, attributed to lac-tate complexed with cations. In this region, there were no signals due to protons of termi-nal methyl groups. No signal due to aromatic proton appeared in either spectra.

Fourier-Transform Infrared Spectroscopy

The DRIFT spectra are shown in Fig. 4. The major spectra bands were assigned as fol-lows: a broad band at around 3200 cm−1 was attributed to O–H stretching of carboxylic and alcoholic groups in different electrostatic

Fig. 1. The 1H nuclear magnetic resonance spectra (8.0–0.6 ppm) of grass humic substance separated into fractions corresponding to high (HMWG) and low (LMWG) relative molecular mass. The peaks are attri-buted according to Fan (1996).

Fig. 2. Region of 1H nuclear magnetic resonance spectra (4.0–2.0 ppm range) of a forest humic substance. The humic substance was separated into fractions with high (HMWF) and low (LMWF) relative molecular mass. The peaks are attributed according to Fan (1996).

SSSAJ: Volume 71: Number 1 • January –February 2007 79

environments (Bellamy, 1975); the band at around 2940 cm−1 was assigned to asymmetric C–H stretching of aliphatic groups. The band appearing at 1719 cm−1 is characteristic of undis-sociated carboxyl groups ν(C=O) vibrations (Rao, 1963; Niemeyer et al., 1992). The bands at 1660 and around 1514 cm−1 probaby correspond to carbonyl C=O stretching and both N–H defor-mation and C–N stretching vibrations. Moreover, these bands are due to C=C and C–C vibra-tions in aromatic rings, respec-tively. The region at around 1400 to 1300 cm−1 correspond to CH2 asymmetric bending and carboxyl-ate symmetric stretching motions (Bellamy, 1975; Niemeyer et al., 1992; Stevenson, 1994); the band appearing at around 1230 cm−1 can be attributed to C–O stretch vibrations in alcohols, phenols, and carboxyl groups. The strong band appearing at around 1043 cm−1 was attributed to C–O stretching of carbohydrates and alco-hols (Bellamy, 1975; Stevenson, 1994), as well as to C–C stretch-ing motions of aliphatic groups, and in-plane C–H bending of aromatic rings.

Comparison of the results in Fig. 4 indicates that there were no signifi cant differences between the two humic frac-tions from grass. The broad band at 2626 cm−1 suggested the formation of intermolecular H bonding between OH groups in oxygenated compounds (Rao, 1963). In addition, the intense bands at around 1718 and 1223 cm−1 confi rmed the presence of COOH groups in acid dimers (Rao, 1963). These data were supported by NMR spectra that showed an intense chemical shift of the β-CH3 protons assigned to lactate (Fig. 1).

The spectrum of humic fractions from forest showed different spectroscopic features assigned to different relative intensities due to COOH and COO− asymmetric stretch-ing. The band at about 1400 cm−1 was identifi ed as typical of monocarboxylic acids (Rao, 1963). The bands associated with acidic groups were identifi ed in the 1H NMR spectrum (Fig. 2), such as acetate and citrate, while lactate was present in lower concentration with respect to the humic fractions from grass (LMWG). Other characteristic bands that appeared in the LMWF spectrum are due to the NH3 deformation at about 1540 cm−1 and the strong band at 1046 cm−1 assigned to C–C skeletal and C–N stretch vibrations. The CH stretching fre-quency at 3084 cm−1 might be linked to methyl groups con-nected with the N atom. In addition, the appearance of proton chemical shift at 3.24 ppm, corresponding to N–CH3 (Fig. 2), was assigned to glycinebetaine.

Callus GrowthCallus incubated on basal medium without growth regula-

tors or humic fractions (control) had a slow growth after 28 d of

Fig. 3. Region of 1H nuclear magnetic resonance spectra (2.0–0.6 ppm range) of high (HMWF) and low (LMWF) relative molecular mass fractions. The peaks are attributed according to Fan (1996).

Fig. 4. Diffuse refl ectance infrared Fourier-transform spectra of a humic substance separated in high (HMW) and low (LMW) relative molecular mass fractions. Humic substances (top) from grass are LMWG and HMWG and (bottom) from forest land are LMWF and HMWF.

80 SSSAJ: Volume 71: Number 1 • January –February 2007

subculture, reaching a weight of 2.41 g. A similar behavior was observed when IAA or BAP were added, but 2,4-D or 2,4-D plus BAP signifi cantly increased the average mass of callus (Table 1). Exposure of P. nigra callus for 28 d to HMWF, LMWF, HMWF plus BAP, and LMWF plus BAP caused a signifi cant decrease of callus growth compared with the other treatments (Table 1) and the callus tissue appeared totally brown and dying. A slight increase of callus growth was observed when HMWF plus IAA or LMWF plus IAA were used. The contemporaneous presence of HMWF or LMWF and 2,4-D or 2,4-D plus BAP signifi cantly increased callus growth (Table 1), and produced callus with less visible brown areas than that grown with HMWF or LMWF.

The presence in medium culture of high- and low-relative-molecular-mass humic fractions derived from grass improved the callus growth with respect to forest humic fractions. Only the combination of grass humic fractions and IAA caused a slight decrease of callus growth (Table 1). In all treatments, the callus tissue appeared white and vigorous.

Carbohydrate ContentAfter 28 d of subculture, 2,4-D alone or in combination

with BAP reduced the amount of hexoses compared with the con-trol callus (Table 2). If treated with either forest humic fraction, a higher content of glucose and fructose in the callus tissue was observed compared with the other treatments. The combination of HMWF or LMWF and 2,4-D or 2,4-D plus BAP strongly decreased the amount of glucose and fructose in callus tissue with respect to that treated with humic fractions alone or in combina-tion with BAP or IAA. In callus tissue grown with grass humic fractions alone or in combination with hormones, glucose and fructose contents were lower than that detected in the callus con-trol or in callus grown with BAP or IAA, and was similar to that found in callus tissue treated with 2,4-D (Table 2).

Amino Acid ContentAfter 28 d of subculture, 2,4-D alone or in combination with

BAP increased the amount of free amino acid compared with the control callus (Table 2). If treated with either forest humic frac-tion, a lower content of amino acid was observed compared with the other treatments. The combination of HMWF or LMWF and 2,4-D or 2,4-D plus BAP increased the amount of amino acids in callus tissue with respect to that treated with humic frac-tions alone or in combination with BAP or IAA. In callus tissue grown with grass humic fractions alone or in combination with hormones, amino acid content was higher than that detected in the callus control or in callus grown with BAP, IAA, or forest humic fractions, and it was similar to that found in callus tissue treated with 2,4-D (Table 2).

Enzyme ActivitiesIn Table 3, the specifi c SAI activity of the different cal-

lus treatments is reported. Both forest humic fractions caused a signifi cant increase of soluble acid invertase compared with control and hormone treatments. The contemporaneous pres-ence of forest humic fractions and 2,4-D or 2,4-D plus BAP decreased the amount of SAI and the values were similar to those detected in callus grown with hormones alone. Instead, in callus tissue treated with forest humic fractions and BAP or IAA, specifi c SAI activity was similar to that observed in callus grown with HMWF or LMWF alone. In callus treated with grass humic fractions alone or in combination with hormones, the values of SAI were similar to those observed in callus tissue grown with plant growth regulators, except IAA.

In callus treated with HMWF or LMWF alone or in com-bination with BAP, the GK activity was lower than the other treatments, showing an inhibition of about 30% with respect to the control and hormone treatments (Table 3). Instead, HMWG or LMWG alone or in combination with plant growth regula-tors did not provoke modifi cations in the GK activity compared with control and hormone treatments (Table 3).

The highest levels of specifi c PGI activity were observed in callus treated with 2,4-D, BAP, or both, with values of 70.18, 75.85, and 71.85 mkat kg−1 protein, respectively (Table 3). Exposure of callus to HMWF or LMWF fractions caused a strong decrease of specifi c PGI activity up to values of 1.73 and 4.16 mkat kg−1 protein, respectively. The combination of HMWF or LMWF with 2,4-D or 2,4-D plus BAP increased the levels of this enzyme (Table 3). When the callus was sup-

Table 1. Biomas and relative growth rate (RGR) of Pinus nigra callus exposed to different hormones and forest or grass humic fractions after 28 d of subculture.

Treatment† Biomass RGR

g d−1

Control 2.41 g‡ 0.34

BAP 2.25 gh 0.22

IAA 2.40 g 0.34

2,4-D 4.00 b 1.10

2,4-D + BAP 5.50 a 1.50

LMWF 1.10 l 0.00

HMWF 1.04 l 0.00

BAP + LMWF 1.18 l 0.00

BAP + HMWF 1.08 l 0.00

IAA + LMWF 2.10 h 0.79

IAA + HMWF 1.90 i 0.67

2,4-D + LMWF 2.89 f 0.64

2,4-D + HMWF 3.30 e 0.82

2,4-D + BAP + LMWF 2.90 f 0.64

2,4-D + BAP + HMWF 3.20 e 0.79

LMWG 3.90 b 1.06

HMWG 3.90 b 1.06

BAP + LMWG 4.20 b 1.16

BAP + HMWG 3.92 b 0.96

IAA + LMWG 3.30 e 0.83

IAA + HMWG 3.10 e 0.74

2,4-D + LMWG 3.80 c 1.03

2,4-D + HMWG 4.00 b 1.10

2,4-D + BAP + LMWG 3.60 d 0.962,4-D + BAP + HMWG 3.50 d 0.92

† Control = callus on MS medium; 2,4-D = 2,4-dichloro phen-oxyacetic acid; BAP = 6 benzylaminopurine; IAA = indole-3-acetic acid; LMWF = low relative molecular mass humic fraction from forest; HMWF = high relative molecular mass humic fraction from forest; LMWG = low relative molecular mass humic fraction from grass; HMWG = high relative molecular mass humic fraction from grass.

‡ Values in the same column followed by the same letter are not statistically different at P = 0.05.

SSSAJ: Volume 71: Number 1 • January –February 2007 81

plemented with HMWF or LMWF plus BAP or IAA, the PGI activity was lower than the control or hormone treatments, but higher than forest HS alone. Exposure of callus tissue to grass humic fractions alone or in combination with hormones increased the PGI activity compared with the control (Table 3).

In callus grown with forest humic fractions, the specifi c ALD activity was low, with values of about 3 mkat kg−1 pro-tein, showing an inhibition of about 70% with respect to hormones. When the callus tissue was treated with HMWF or LMWF and BAP, the specifi c ALD activity was low: 4.50 and 4.85 mkat kg−1 protein, respectively. Instead, when the callus was grown with forest humic fractions and 2,4-D or 2,4-D plus BAP or IAA, the ALD activity strongly increased, reaching values similar to those observed in callus grown with hormones (Table 3). In callus grown for 28 d on HMWG or LMWG alone or in combination with hormones, the ALD level increased compared with the control and it was similar to that detected in callus treated with hormones alone (Table 3).

The PK activity was lower in callus treated with HMWF or LMWF alone or in combination with BAP compared with the other treatments (Table 3). The callus grown with grass humic fractions alone did not show signifi cant changes in PK activity with respect to control, 2,4-D, BAP, and IAA, but it was lower with respect to 2,4-D plus BAP (Table 3).

The GS activity was inhibited by the presence in the medium culture of HMWF or LMWF alone or in com-bination with BAP compared with the other treatments (Table 4). In callus grown in the presence of grass humic fractions alone or in combination with 2,4-D or 2,4-D plus BAP, the GS levels were similar to that detected in callus treated with 2,4-D plus BAP (Table 4).

The GOGAT activity did not show signifi cant changes in the different treatments (Table 4).

The GDH activity was lower in callus treated with forest humic fractions alone or with BAP, while it was increased by HMWF or LMWF and 2,4-D or 2,4-D plus BAP (Table 4). In the presence of grass humic fractions alone or in combination with 2,4-D, the GDH activity was similar to that detected in the control or in callus grown with 2,4-D, but it was lower than that found in callus treated with 2,4-D plus BAP alone or in combina-tion with HMWG or LMWG (Table 4).

Both forest humic fractions alone or with BAP caused a decrease of MDH activity with respect to the other treatments (Table 4). Instead, in the presence of forest humic fractions and 2,4-D or 2,4-D plus BAP, it was possible to observe a strong increase in MDH activity compared with control (Table 4). When the grass humic fractions were added to medium culture, they strongly increased the MDH activity in callus compared with the control, IAA, or BAP, and the values were similar to that detected in callus treated with 2,4-D alone or in combi-nation with grass humic fractions (Table 4).

In the control or in callus grown in the presence of IAA or BAP, the PEPC levels signifi cantly decreased with respect to 2,4-D or 2,4-D plus BAP (Table 4). In callus grown with forest humic fractions alone or in combination with BAP or IAA, it was possible to observe a lack of this activity. On the contrary, in the presence of forest humic fractions and

2,4-D or 2,4-D plus BAP, the PEPC activity increased, reaching values similar to those observed in callus treated with 2,4-D plus BAP (Table 4). When the callus was supplemented with HMWG or LMWG alone or in combination with hormones, the PEPC levels were similar to that determined in callus treated with 2,4-D plus BAP (81.68 mkat kg−1 protein) and this value was higher than that determined in the control or in callus grown with IAA or BAP (Table 4).

DISCUSSIONMainly, we have investigated the effects of HS on the C and

N metabolism of callus, since callus grown in vitro does not have a photosynthetic activity and uses the sucrose of the medium culture as a C source (Dukema et al., 1988). The use of sucrose is necessary to provide C for respiration processes, and, in most investigated species, it is rapidly hydrolyzed to glucose and fruc-tose before use. Sucrose may be cleaved either by SuSy (sucrose synthase), a cytosolic enzyme that is crucial to sucrose utilization in fruit development (Sun et al., 1992; Wang et al., 1993), and SAI, a hydrolase that is particularly active in rapidly growing tis-sues (Faye and Ghorbel, 1983; Arai et al., 1991). The invertase levels in callus treated with either forest humic fraction were sig-

Table 2. Sucrose, glucose, fructose (g L−1), and free amino acid (f.w.) content in Pinus nigra callus grown for 28 d with hormones and with or without forest or grass humic fractions.

Treatment† Sucrose Glucose Fructose Amino acid

——————— g L−1 ————— g kg−1

Control 0.33 f‡ 4.14 cd 10.90 c 0.73 f

BAP 1.97 a 3.80 d 10.60 c 0.69 g

IAA 0.31 g 4.92 c 10.90 c 0.74 f

2,4-D 0.82 b 1.90 e 3.47 f 1.30 c

2,4-D + BAP 0.33 fg 1.46 f 2.95 g 1.41 b

LMWF 0.60 c 10.45 a 13.95 ab 0.32 m

HMWF 0.22 h 11.33 a 14.50 a 0.33 m

BAP + LMWF 0.45 d 10.08 a 14.02 ab 0.39 i

BAP + HMWF 0.65 c 10.45 a 14.95 a 0.44 h

IAA + LMWF 0.31 g 7.40 b 13.35 b 0.35 l

IAA + HMWF 0.40 de 7.40 b 13.90 ab 0.29 n

2,4-D + LMWF 0.30 g 5.80 c 4.50 d 1.01 e

2,4-D + HMWF 0.33 fg 5.95 c 4.65 d 1.00 e

2,4-D + BAP + LMWF 0.75 b 2.01 e 3.85 e 1.09 e

2,4-D + BAP + HMWF 0.35 f 1.81 e 3.39 f 1.11 e

LMWG 0.35 f 1.96 e 3.70 e 1.35 bc

HMWG 0.45 d 1.93 e 3.90 e 1.29 c

BAP + LMWG 0.35 f 1.45 f 3.55 ef 1.34 bc

BAP + HMWG 0.36 ef 1.59 f 3.47 f 1.21 d

IAA + LMWG 0.37 e 2.10 e 4.35 d 1.31 bc

IAA + HMWG 0.34 f 2.25 e 4.47 d 1.44 ab

2,4-D + LMWG 0.43 d 1.74 ef 3.50 ef 1.51a

2,4-D + HMWG 0.33 fg 1.69 f 3.62 ef 1.49 a

2,4-D + BAP + LMWG 0.39 e 1.60 f 3.95 e 1.45 ab2,4-D + BAP + HMWG 0.38 e 1.64 f 3.87 e 1.49 a

† Control = callus on MS medium; 2,4-D = 2,4-dichlorophenoxyacetic acid; BAP = 6 benzylaminopurine; IAA = indole-3-acetic acid; LMWF = low molecular weight humic fraction from forest; HMWF = high molecular weight humic fraction from forest; LMWG = low molecular weight humic fraction from grass; HMWG = high molecular weight humic fraction from grass.

‡ Values in the same column, followed by the same letter are not statistically different at P = 0.05.

82 SSSAJ: Volume 71: Number 1 • January –February 2007

nifi cantly increased with respect to the other treatments, show-ing that the invertase activity and the hydrolysis of sucrose into glucose and fructose were not affected. The products of sucrose cleavage are then converted to hexoses phosphates, and in this way they can enter into the respiratory pathway, via glycolysis, to pro-vide substrates and reducing power for growth. We have found an accumulation of the two hexoses in callus tissue treated with these two humic fractions, suggesting a block of the glycolytic pathway. In fact, the activity of glucokinase, the enzyme that converted the products of sucrose cleavage to hexoses phosphates (Dennis and Greyson, 1987; Kruger, 1990; Galina et al., 1999), was lower in callus treated with either forest humic fraction, indicating an inter-ference of these forest humic fractions in the glycolytic pathway. The PGI, in a subsequent step of glycolysis, catalyzes the conver-sion of glucose-6-phosphate to fructose-6-phosphate (Appeldoorn et al., 1977). The activity of this enzyme was strongly inhibited in P. nigra callus in the presence of either forest humic fraction, indi-cating that PGI is the enzyme whose activity is most affected by these fractions. The inhibition of PGI caused signifi cant declines in the activity of ALD, the enzyme involved in the conversion of hexoses phosphates to triose phosphates (Ashihara et al., 1988). Aldolase declined in callus treated with forest humic fractions, and the same behavior was observed in the activity of PK, the enzyme

considered a major metabolic intersec-tion linking the glycolytic path to the Krebs cycle (Baysdorfer and Bassham, 1984; Geigenberger and Stitt, 1991).

One important level of competi-tion between C and N metabolism is related to C chains that accept reduced N. The synthesis of amino acids interacts with the Krebs cycle, which provides a C skeleton for glutamate and aspartate (Alisdair et al., 2004). Different researchers have demon-strated an increase in amino acids with a simultaneous decrease in carbohy-drate when NH4

+ (Platt et al., 1977) or NO3

− (Champigny and Foyer, 1992; Amancio et al., 1993) is supplied.

Thus, the drastic decrease of callus growth in the presence of for-est HS is a consequence of the strong inhibition of PGI activity, which consequently causes a lower use of glucose and fructose in callus tissue, damage to C metabolism, and also an inhibition of PEPC, GDH, MDH, and GS, enzymes that provide a link between carbohydrate and amino acid metabolism (Stulen, 1986; Copeland et al., 1989; Robinson et al., 1992; Raab and Terry, 1995). These effects were reversed when the forest frac-tions were used concomitantly with 2,4-D or 2,4-D plus BAP, they were only partially reversed when used with IAA, but they were not reversed in the presence of BAP alone, suggesting that 2,4-D is the hormone that avoids, in

the best way, the negative effect of these fractions on callus tissue. The fi ndings that 2,4-D is better than IAA to reverse the nega-tive effects of humic fractions from forest soil could be explained by the different roles that every auxin has on callus induction for various species (Tao et al., 2002).

An opposite behavior was observed when the callus was grown in the presence of grass humic fractions; in fact, they are capable of improving the growth of callus by increasing the levels of the activ-ity of the key enzymes involved in C and N metabolism.

All the data support the view that the biological activity of HS utilized is independent of their molecular weight, since both fractions (high and low relative molecular mass) obtained from the same HS have a similar behavior on callus tissue.

Humic substances are considered as a sort of memory of microbial population and plant cover, where the active ingredients are not mineral nutrients, but organic acids and biologically active metabolites of various microbes; thus, as reported by Frankenberger and Arshad (1995), HS derived from soils with different plant cover may have a different chemical composition.

The results of 1H-NMR spectra and the DRIFT spectros-copy confi rmed this, showing signifi cant differences in the chem-ical structure between forest and grass HS. A great amount of

Table 3. Specifi c activity of key C metabolism enzymes (soluble acid invertase [SAI], gluco kinase [GK], phosphoglucoisomerase [PGI], aldolase [ALD], and pyruvate kinase [PK]) in Pinus nigra callus grown for 28 d with hormones and with or without forest or grass humic fractions.

Treatment† SAI GK PGI ALD PK

mkat kg−1 protein μkat kg−1 protein ———— mkat kg−1 protein ———

Control 5.00 h‡ 13.3 b 50.00 c 7.16 c 11.17 b

BAP 15.00 de 13.6 b 75.85 a 11.33 a 10.83 b

IAA 28.33 c 13.3 b 67.01 b 10.83 a 10.50 c

2,4-D 7.33 g 13.8 b 70.18 a 11.67 a 11.17 b

2,4-D + BAP 4.66 h 15.0 a 71.85 a 11.50 a 13.84 a

LMWF 140.70 b 10.8 c 4.16 g 2.83 e 3.50 e

HMWF 180.04 a 10.1 c 1.73 g 3.33 e 3.83 e

BAP + LMWF 140.86 b 10.5 c 17.00 e 4.50 d 4.67 e

BAP + HMWF 166.70 a 10.0 c 12.16 f 4.83 d 4.17 e

IAA + LMWF 132.36 b 13.3 b 32.50 d 10.00 b 7.97 d

IAA + HMWF 130.03 b 13.8 b 35.00 d 10.50 b 7.67 d

2,4-D + LMWF 5.84 h 14.8 a 62.18 b 10.50 b 10.17 c

2,4-D + HMWF 10.84 f 13.3 b 65.68 b 10.83 a 9.67 c

2,4-D + BAP + LMWF 4.67 h 15.2 a 63.01 b 10.83 a 14.34 a

2,4-D + BAP + HMWF 5.84 h 15.7 a 63.35 b 11.17 a 14.17 a

LMWG 18.34 d 14.2 a 65.85 b 11.33 a 12.00 b

HMWG 15.84 d 14.5 a 64.85 b 10.67 b 11.84 b

BAP + LMWG 15.17 de 14.2 a 62.68 b 10.83 a 10.34 c

BAP + HMWG 14.17 e 14.3 a 63.51 b 10.67 b 10.17 c

IAA + LMWG 15.17 de 13.7 b 67.01 b 10.50 b 10.67 bc

IAA + HMWG 8.17 g 13.5 b 68.85 a 10.67 b 10.50 c

2,4-D + LMWG 7.50 g 14.5 a 65.86 b 11.17 a 11.35 b

2,4-D + HMWG 5.84 h 14.8 a 64.85 b 11.00 a 11.32 b

2,4-D + BAP + LMWG 15.17 de 15.0 a 69.18 a 11.50 a 11.33 b2,4D + BAP + HMWG 14.34 e 14.8 a 70.51 a 11.33 a 11.25 b

† Control = callus on MS medium; 2,4-D = 2,4-dichlorophenoxyacetic acid; BAP = 6 benzy lamino purine; IAA = indole-3-acetic acid; LMWF = low relative molecular mass humic fraction from forest; HMWF = high relative molecular mass humic fraction from forest; LMWG = low relative molecular mass humic fraction from grass; HMWG = high relative molecular humic fraction from grass.

‡ Values in the same column, followed by the same letter are not statistically different at P = 0.05.

SSSAJ: Volume 71: Number 1 • January –February 2007 83

aliphatic and H-sugarlike com-ponent and an intense chemical shift of the β-CH3 region were observed in both grass humic fractions, while high contents of betaine, organic acids, and COOH groups were detected in both forest humic fractions.

In conclusion, this study shows that the parental organic materials may comprise com-pounds that serve as precursors or substrates for the synthesis of biologically active substances by the heterotrophic activity of soil biota, and, at the same time, gives evidence that the chemical structure of HS is well refl ected in their biological activity.

As HS have very complex structures, it is very diffi cult to identify the relationship between the single compounds of HS and their biological activity. Therefore more research is nec-essary to explain the effects of humus, focusing attention on a much more detailed separation and analysis of these fractions, with a better chemical identifi ca-tion, and subsequent testing of pure compounds.

ACKNOWLEDGMENTSThis research was supported by the Università degli Studi Mediterranea di Reggio Calabria—Programmi di Ricerca Scientifi ca (ex Quota 60%). The authors thank Ms. A. Cummins for revising the language.

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Table 4. Specifi c activity of N metabolism enzymes (glutamine synthetase [GS], glutamate synthase [GOGAT], glutamate dehyrogenase [GDH], phosphoenolpyruvate carboxylase [PEPC] and malate dehydrogenase [MDH]) in Pinus nigra callus grown for 28 d with hormones and with or without forest or grass humic fractions.

Treatment† GS GOGAT (10-2) GDH MDH PEPC

μkat kg−1 protein mkat kg−1 protein μkat kg−1 protein mkat kg−1 protein

Control 2.71 e‡ 0.41 b 1.23 d 0.83 e 14.67 f

BAP 3.00 d 0.34 c 1.00 e 1.17 d 17.50 e

IAA 2.90 d 0.39 b 0.46 f 1.00 d 10.53 g

2,4-D 4.30 a 0.36 c 1.25 d 2.83 c 108.35 a

2,4-D + BAP 3.50 b 0.45 a 2.33 a 3.12 b 81.68 b

LMWF 2.16 f 0.37 bc 0.17 h 0.17 f ND§

HMWF 2.08 f 0.37 bc 0.28 g 0.12 f ND

BAP + LMWF 2.15 f 0.37 bc 0.32 g 0.23 f ND

BAP + HMWF 2.10 f 0.37 bc 0.25 g 0.17 f ND

IAA + LMWF 2.97 d 0.39 b 1.08 e 0.58 e 5.83 h

IAA + HMWF 2.50 e 0.39 b 0.33 g 2.84 c 3.83 i

2,4-D + LMWF 3.85 b 0.37 bc 1.33 d 2.84 c 85.85 b

2,4-D + HMWF 4.00 ab 0.36 c 1.16 e 3.33 a 76.51 c

2,4-D + BAP + LMWF 3.48 b 0.38 b 1.58 c 3.00 b 70.84 d

2,4-D + BAP + HMWF 3.67 b 0.41 b 1.63 c 3.53 a 71.34 d

LMWG 3.48 bc 0.36 c 1.21 d 2.67 c 80.85 b

HMWG 3.50 b 0.37 bc 1.25 d 2.75 c 81.85 b

BAP + LMWG 3.32 c 0.36 c 1.17 e 3.08 b 78.35bc

BAP + HMWG 3.30 c 0.37 bc 1.17 e 3.08 b 81.68 b

IAA + LMWG 3.10 cd 0.39 b 1.07e 1.25 d 70.01 d

IAA + HMWG 3.13 c 0.36 c 1.08 e 1.32 d 73.35 c

2,4-D + LMWG 3.67 b 0.40 b 1.23 d 2.84 c 83.35 b

2,4-D + HMWG 3.68 b 0.39 b 1.22 d 2.86 c 85.01 b

2,4-D + BAP + LMWG 3.47 b 0.41 b 1.82 b 3.17 b 68.34 d2,4-D + BAP + HMWG 3.40 b 0.40 b 1.97 b 3.12 b 70.01 d

† Control = callus on MS medium; 2,4-D = 2,4-dichlorophenoxyacetic acid; BAP = 6 benzylaminopurine; IAA = indole-3-acetic acid; LMWF = low relative molecular mass humic fraction from forest; HMWF = high relative molecular mass humic fraction from forest; LMWG = low relative molecular mass humic fraction from grass; HMWG = high relative molecular mass humic fraction from grass.

‡ Values in the same column, followed by the same letter are not statistically different at P = 0.05.

§ ND = not detectable.

84 SSSAJ: Volume 71: Number 1 • January –February 2007

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