Stress Responses in Alfalfa (Medicago sativa L.) : V. Constitutive and Elicitor-Induced Accumulation of Isoflavonoid Conjugates in Cell Suspension Cultures

The isoflavonoid conjugates medicarpin-3-O-glucoside-6″-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6″-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with [¹⁴C]phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, [¹⁴C]phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of ¹⁴C-labeled, elicited cells with l-α-aminooxy-β-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures.     

The establishment of cell suspension culture of sabah snake grass (<Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Burm.F.) Lindau)

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L^?1 2,4-D and 0.75 mg L^?1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L^?1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L^?1 2,4-D and 0.50 mg L^?1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Characteristics of galacturonic Acid oligomers as elicitors of casbene synthetase activity in castor bean seedlings

The metabolism of [¹⁴CH₃]2-(2,4-dichlorophenoxy)isobutyric acid (DIB) was studied in plants and cell suspension cultures of Lycopersicon esculentum Mill. sp. `Lukullus'. Both plants and cells in suspension culture showed a rapid uptake of DIB from nutrient media. The metabolites, isolated by extraction with methanol and separated by chromatographic methods, were identified by enzymic, chemical, and spectrometric methods. Two conjugates of the carboxyl with 2 and 3 moles glucose per mole DIB and, to a smaller extent, its β-d-glucopyranosyl ester, were formed in both intact plants and cell suspension cultures, but there were quantitative differences.     

Production of human lactoferrin in transgenic cell suspension cultures of sweet potato

Shoot apical meristem-derived calli were transformed with a hLF cDNA in an attempt to produce human lactoferrin (hLF) in transgenic cell suspension cultures of sweet potato [Ipomoea batatas (L.) Lam.]. Calli were bombarded with tungsten particles coated with the binary vector pLSM1 containing a hLF cDNA under the control of the 35S promoter and the neomycin phosphotransferase gene as a selection marker. Calli were then transferred to Murashige and Skoog (MS) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 100 mg dm⁻³ kanamycin. Kanamycin-resistant calli were selected at four-week intervals and subcultured. Cell suspension cultures were established in liquid MS medium with 4.52 μM 2,4-D. Southern and Northern blot analyses confirmed that hLF cDNA was incorporated into the plant genome and was properly expressed in the cells. ELISA analysis showed that transgenic cells produced hLF up to 3.2 μg mg⁻¹ (total protein).     

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l^-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l^-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth and triterpenic acid accumulation of Cyclocarya paliurus cell suspension cultures

Cyclocarya paliurus is a unique plant growing in central China with hypoglycaemic and hypolipaemia effects. To make better use of this functional food resource, cell suspension cultures and triterpenic acid accumulation were studied. Stable and uniform cell suspension cultures were established in liquid basal Murashige and Skoog medium supplemented with 2,4-dichlorophenoxy acetic acid (0.5 mg/L), naphthalene acetic acid (0.3 mg/L) and cytokinin (1.0 mg/L). According to the growth curve and triterpenic acid accumulation curve, the 8 ～ 10^th day postinoculation was the optimum time for subculture, and the 14^th day was the optimum time for harvest. Murashige and Skoog medium and woody plant medium were suitable for both cell growth and triterpenic acid accumulation. 3% sucrose (w/v), 60 mM total nitrogen (NO₃ ^?/NH₄ ⁺ = 2/1), 1.25 mM KH₂PO₄, 2 mM CaCl₂, and 2 mM MgSO₄ were all found to be fit for cell growth and triterpenic acid accumulation in a cell suspension culture of Cyclocarya paliurus. Total triterpenic acid, ursolic acid and oleanolic acid content in suspended cultured cells were all significantly higher than that of leaves and calluses (P ? 0.01), with levels up to 6.24, 2.28, and 0.94% (of dry weight), respectively. The betulinic acid content of suspended cultured cells also reached 0.82%, which was significantly higher than that of calluses. These results suggest that suspended cultured cells of Cyclocarya paliurus were rich in triterpenic acids and could be used for the production of total triterpenic acid, ursolic acid, oleanolic acid and betulinic acid.     

Higher Production of Forskolin in Genetically Transformed Cultures of Coleus forskohlii Briq Induced by Growth Regulators

Increased forskolin yield was obtained in transformed root, rhizogenic calli and cell suspension cultures of Coleus forskohlii when treated alone with various concentrations of auxins (IAA, IBA, NAA, 2,4-0), auxin conjugates ( IAA-ala, IAA-gly, IAA-phe, IAA-asp), cytokinins (Kn, BAP) and gibberellic acid (GA₃). An 8.9-fold stimulation in forskolin production was achieved in transformed rhizogenic line GCO-RCH-10 in presence of 1.0 mg I^-1 GA₃, 6-fold in cell suspension line GSO-5/7-k in presence of 2 mg I^-1 BAP and 4.3-fold in root line RC-ST -2/16 in presence of 0.5 mg I^-1 GA₃ at the end of a culture period of 4 weeks. Growth and morphology was found to be influenced by the growth regulators studied. A seven fold increase in biomass was obtained in rhizogenic line GCO-RCH-10 with 0.5 mg I^-1 GA₃ In root line RC-ST-2/16, different concentrations of IAA, IAA conjugates and GA3 stimulated growth while cytokinins inhibited growth of roots. The shoot culture line ST -2/51/d, showed prolific growth in the presence of all cytokinins but no forskolin was detected in the shoot cultures treated with any of these hormones.     

Auxin requirements of sycamore cells in suspension culture

Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this `auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.

There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10⁶ cells) on day 2 followed by a decline to 45 nanograms per 10⁶ cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10⁶ cells) rises slowly to a peak of 1.4 nanograms per 10⁶ cells by day 9 then decreases rapidly to 0.2 nanograms per 10⁶ cells by day 15 (early declining phase) and 0.08 nanograms per 10⁶ cells by day 23 (early stationary phase).

     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Translocation and Complex Formation of Picloram and 2,4-D in Wheat Seedlings

Uptake, translocation and metabolism of ¹⁴C-labelled 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) in seedlings of wheat (Triticum aestivum L.) were studied. The uptake of the herbicides through the upper surface of the first leaf was slow but was almost complete after nine days. Picloram was absorbed faster than 2,4-D. Picloram was also translocated into the stem and the untreated leaves to a greater extent than 2,4-D. Only small fractions of the activity were recovered from the roots and from the nutrient solution. Picloram and 2,4-D formed water-soluble conjugates in the tissues. These conjugates were very labile and hydrolyzed under release of the unchanged herbicides. The isotope from 2,4-D was also incorporated in an insoluble fraction, containing cell walls and proteins. Also from this fraction biologically active 2,4-D could be released by hydrolysis. The formation of the complexes was partly prevented by cycloheximide. It is suggested that herbicide detoxification through complex formation is of importance for the relatively low sensitivity of wheat to auxin herbicides.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

The effect of ionizing irradiation on the tissue culture ofCoronilla varia

Long-term callus cultures of crownvetch (Coronilla varia L.) grown on the Murashige and Skoog’s medium with 2,4-D (1 mg 1^-1) and cultures of somatic embryos cultivated on the same basic medium but with IAA (1.0 mg I^-1) were exposed to ionizing irradiation. The irradiation caused a growth inhibition excepting the lowest dose of 2.5 Gy. The highest dose of 160 Gy induced browning of the culture but this colour change was not lethal. The amount of “giant cells” present in both cultures was dependent on the dose of irradiation.     

Obtaining and Study of Callus and Suspension Plant Cell Cultures of <Emphasis Type="Italic">Tribulus terrestris</Emphasis> L., a Producer of Steroidal Glycosides

Callus and suspension plant cell cultures of Tribulus terrestris L., a valuable medicinal plant producing steroidal glycosides, were obtained. The seeds from an American population of T. terrestris were used as explants. Regulation of the production and growth of cell cultures, as well as the biosynthetic characteristics of the cell lines, were studied. The combination of phytohormones of 2,4-D (2.0 mg/L) and BAP (1.0 mg/L) was found to be optimal for callus induction and cultivation. Suspension cell culture obtained in liquid medium of the same composition showed such high growth characteristics during prolonged cultivation (more than 2 years) as a maximum accumulation of dry biomass of 13 g/L, specific growth rate at exponential phase of 0.24 day^–1, and economical coefficient of 0.39. A semicontinuous mode of cultivation was used to grow the plant cell suspension in a lab-scale bioreactor. Screening of the steroidal glycosides in the obtained cell cultures was carried out. Steroidal glycosides were not found in the callus cultures. However, as was demonstrated by TLC and UPLC ESI MS methods, the suspension culture contained furostanol glycosides, and their amount increased during the cultivation process. These results support the hypothesis of the autoselection of cultivated cells containing compounds promoting their proliferation in vitro.     

Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - Ethrel 2-chlorethylphosphonic acid - NAA 1-naphthaleneacetic acid - NR^– nitrate reductase deficient     

Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10⁴- 1×10⁵ protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - Kin 6-Furfurylaminopurine (kinetin) - NAA -Naphtalenacetic acid - Zea Zeatin     

Substrate Utilization by Suspension Cultures and Somatic Embryos of Daucus carota L. Measured by C NMR

The uptake and utilization of sucrose by embryogenic suspension cultures of carrot (Daucus carota L.) growing in the presence of 2,4-D and by somatic embryos derived from these cultures was monitored using ¹³C nuclear magnetic resonance. The exogeneously supplied sucrose was completely hydrolyzed before cell entry; glucose was taken up preferentially when the cells were cultured in the presence of 2,4-D, while glucose and fructose were utilized at similar rates by somatic embryos in the absence of 2,4-D. Both suspension cells and somatic embryos accumulated high intracellular levels predominantly of glucose and sucrose, the latter being resynthesized intracellularly from the constitutive hexoses. Initially, fructose was converted mainly into glucose and sucrose rather than being catabolized directly through glycolysis or the pentose phosphate pathway. Carbohydrate supply that exceeded cellular demand resulted in intracellular accumulation of mono- or disaccharides. The capacity of cultured carrot cells to produce somatic embryos appeared to be positively correlated with high intracellular levels of glucose.     

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.