Activity of antioxidant enzymes and secondary metabolites during in vitro regeneration of Sterculia urens

The changes in the activities of antioxidant enzymes and amounts of proteins, phenols, and flavonoids in regenerating and non-regenerating calli during organogenesis of Sterculia urens were monitored. Maximum growth of calli and the most efficient regeneration of shoots occurred on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^?3 benzylaminopurine (BAP) + 2 or 4 mg dm^?3 naphtalene acetic acid (NAA). Peroxidase (POD), catalase, and superoxide dismutase activities increased in the regenerating calli but decreased in the non-regenerating calli. Six POD isoenzymes were detected. Protein content decreased in the non-regenerating calli and increased significantly during regeneration of shoots from callus. Total phenols and flavonoids increased in the non regenerating calli. SDS-PAGE analysis revealed a role of many proteins in organogenesis.     

The role of roots in sex expression in hemp plants

When the shoots of young hemp (Cannabis sativa L.) plants were cut off the roots, cultured as cuttings, and regenerating (adventitious) roots were removed as soon as appearing, ca. 80–90% of the plants became male (had staminate flowers) whereas if the roots were allowed to develop a similar percentage became female (pistillate flowers). Treatment of de-rooted cuttings with 6-benzylaminopurine (15 mg/l) restored the percent of female plants to ca. 80. It is suggested that the root system plays an essential role in sex expression in hemp and that this role is related to cytokinin synthesis in the root.     

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.     

Quinones as plant growth regulators

The effects of benzoquinone, naphthaquinone and anthraquinone on the growth of tomato callus, whole plants of tomato and on rooting of mungbean cuttings were studied. Naphthaquinone effects on some oxidases and on the isozyme patterns of peroxidases in all the three systems were also observed. Quinones increased callus growth, the number of roots initiated in mungbean cuttings and the growth of tomato plants, significant increases being obtained with 10^–5 M naphthaquinone. Naththaquinone also decreased the activities of IAA oxidase, ascorbic acid oxidase and polyphenol oxidase, and led to the disappearance of one of the isozyme of peroxidase in all systems.     

Veränderungen im oxidativen Stoffwechsel der Tomatenpflanze (Solanum lycopersicum L.) bei Befall mit der Blattrollkrankheit der Kartoffel

Metabolic changes in tomato plants infected with potato leafroll virus were followed. The virus was transferred by grafts taken from diseased tomato plants. Sharp changes in the respiration rate and in the activities of the investigated enzymes observed before the concrescence of the graft with the stock are obviously connected with the defence reactions of the plant. In the roots of the experimental plants a nearly complete correspondence of the course ofo-diphenol oxidase and ascorbic acid oxidase activities with the respiration rate occurs. In the shoots the respiratory chain with ascorbic acid oxidase as terminal oxidase is involved, whereas both chains probably take part in the respiration in the roots by which a link with sugar degradation is achieved. The rate of glycolysis and that of pentose phosphate cycle in the roots of experimental plants are nearly identical. The comparison of glucose-6-phosphate dehydrogenase activity with ascorbic acid oxidase activity reveals that both curves show the same course, which indicates the presence and action of a respiratory thain with ascorbic acid oxidase functioning as terminal oxidase.     

Microcutting leaf area,weight and position on the stock shoot influence root vigour,shoot growth and incidence of shoot tip necrosis in grape plantlets in vitro

The physiological parameters of microcuttings, namely leaf area, weight (wt) and position on the stock shoot, had significant effects on root vigour, microshoot growth and incidence of shoot tip necrosis (STN) in cultures of grape (Vitis vinifera L.) ‘Arka Neelamani’. Single-node leafy cuttings cultured in MS medium containing 1 μM IAA and 0.1 μM GA₃ generally rooted first and subsequently sprouted into a single shoot. Small leafed cuttings exhibited slow root emergence, poor quality roots, early sprouting and weak shoot growth. Large leafed cuttings on the other hand, showed early rooting, vigorous roots, delayed sprouting and healthier shoots. Significant correlations were observed between fresh root wt per plantlet at 1 month in culture and wt of lamina, total wt and leaf area of the cuttings in that order. A significant correlation also existed between wt of roots and height or wt of the sprout that developed. The study suggests that the weight or area of the leaf governed the root growth in a microcutting. STN was observed in some plantlets particularly those derived from large leafed cuttings. Such cuttings showed vigorous roots and delayed but fast sprout growth that ended in STN. Plantlets showing STN had less Ca⁺⁺ and Mg⁺⁺ in the shoot tissue than in the shoots of normal plantlets while the roots showed similar Ca⁺⁺ or higher Mg⁺⁺ contents. Both had comparable amounts of cytokinins in shoot and root tissues. This revised version was published online in June 2006 with corrections to the Cover Date.     

间苯二酚、水杨酸对绿豆下胚轴不定根形成的作用

２０—１００ｍｇＬ（－１）间苯二酚能明显地促进绿豆下胚轴不定根的形成,与２０ｍｇＬ（－１）ＩＢＡ混合处理具加成效应,其作用在于降低生根初期ＩＡＡ氧化酶和多酚氧化酶活性．１０—１００ｍｇＬ（－１）水杨酸抑制下胚轴不定根的形成,随处理浓度的加大,对生根数目、生根范围和根重的抑制作用增加．水杨酸处理后１－３ｄ,能提高ＩＡＡ氧化酶和多酚氧化酶的活性．     

Changes in auxin protectors and IAA oxidases during the rooting of chestnut shoots in vitro

Auxin protectors and IAA oxidase activity were comparatively analyzed in the upper and the lower parts of shoots of chestnut ( Castanea sativa Mill.) cultivated in vitro with indolebutyric acid (IBA) pretreatment. Rhizogenesis of the shoots is accompanied by an increase in auxin protectors in the lower parts and by a decrease of these protectors in the upper parts. Besides, the IAA oxidase activity declines in the basal parts during the rooting process while it increases in the upper ones. These biochemical events would enhance the IAA level in the rooting region of the shoots. In untreated, non-rooted cuttings, the IAA oxidase activity remains low in the upper parts and high in the basal parts of the shoots. The results thus indicate that the IBA treatment may control the endogenous auxin level of the cuttings, either through a direct regulation of the IAA oxidase system or more indirectly through the transport of auxin protectors.     

Polar transport and accumulation of indole-3-acetic acid during root regeneration by Pinus lambertiana embryos

Summary The relation of indoleacetic acid (IAA) transport to accumulation of auxin at the base of cuttings and to polar root formation was investigated with small cuttings from germinating embryos of Pinus lambertiana.The transport of endogenous auxin participates in regeneration of roots. This is shown by the facts that (1) more than 40% of the cuttings rooted without addition of exogenous indoleacetic acid; (2) the first regeneration always occurred at the basal tip of a slanting cut; and (3) 2,3,5-triiodobenzoic acid (TIBA), a specific inhibitor of auxin transport, totally inhibited rooting. Addition of IAA to the medium increased the number of roots formed per rooting hypocotyl.Sections of hypocotyls excised from dormant embryos and tested immediately after 2 h hydration were capable of polar transport of IAA. This polarity increased during the first 3 days of culture because of a marked increase in basipetal transport. Culturing the cuttings in 1 M IAA for 3–5 days doubled both the basipetal transport of 1-¹⁴C-IAA by hypocotyl segments and the accumulation of radioactivity at the base of cuttings.The extent of the accumulation at the base of cuttings was similar at early (2 days, first mitoses) and late stages (5 days, organized meristem) of regeneration and was not affected by removal of the regenerating region immediately prior to uptake and transport of ¹⁴C-IAA. The accumulation was inhibited by TIBA. In terms of increase in wet and dry weight and mitotic activity, the cotyledons rather than the regenerating root meristems were the most actively growing region of the cuttings. The upper part of the hypocotyl elongated more than the region of the slanting cut where regeneration was occurring.These results provide no support for the idea that the regenerating root controls the direction of polar transport by acting as a sink. The results are consistent with the view that polar auxin transport delivers auxin to the base of the cutting and raises the local concentration to levels sufficient to promote root formation.     

Comparison of Cadmium Effect on Willow and Poplar in Response to Different Cultivation Conditions

Salix alba L. and Populus×euroamericana cv. Robusta cuttings were grown in 10 μM Cd(NO₃)₂ (direct treatment) or in Knop solution and afterwards in Cd(NO₃)₂ (indirect treatment). Cd impact on rooting of directly treated plants and its impact on normally formed roots and shoots of indirectly treated plants were studied. The cumulative length, number and biomass of willow roots, pigment and starch contents, leaf net photosynthetic rate and dry mass/leaf area ratio of willow leaves were positively influenced by indirect treatment. However, indirectly treated poplars were more sensitive to Cd than directly treated ones. Indirect treatment lowered root Cd uptake in willow, Cd accumulation in cuttings of both species and Cd accumulation in poplar shoots. Cd-caused structural changes were similar in both species and in both treatments. Root apices, rhizodermis and cortex were the most seriously damaged root parts. In directly treated willow, the structure of central cylinder (0.5 – 1 cm from apex) remained unchanged in contrast to indirectly treated plants. Formation of cambium close to the apex indicated shortening of root elongation zone of indirectly treated plants. Directly Cd-treated poplar roots exhibited unusual defence activity of root apical meristem and accumulation of darkly stained material around central cylinder. This revised version was published online in July 2006 with corrections to the Cover Date.     

Modified hormonal balance in rooting-recalcitrant rac mutant tobacco shoots

ABSTRACT

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the D8 wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurred at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delayed in the basal parts of the rac stems compared to the wild-type ones. The mutant shoots contained higher levels of benzyladenine and isopentenyladenosine at the end of the culture cycle, whereas zeatin riboside was more abundant in wild-type shoots. In response to increasing concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), only the wild-type shoots responded by an increase in growth rate followed by inhibition at high concentrations. The rac shoot responses were very low or nonexistent. Peroxidase activity was also measured in the basal parts of tobacco stems grown in the presence of IBA. Results suggest growth inhibition related to auxin accumulation, possibly combined with elevated putrescine content. Second, rooting induction seems to take place in both tobacco genotypes; however, the process of root formation is blocked in the mutant. The lack of initiation and expression phases of rooting in relation to auxin content in the mutant is discussed.     

Comparative effect of IBA, BSAA and 5,6-Cl2-IAA-Me on the rooting of hypocotyl in mung bean

Mung bean hypocotyl cuttings were treated with indole-3-butyric acid (IBA), 3-(benzo[b]selenienyl)acetic acid (BSAA) and 5,6-dichloroindole-3-acetic acid methyl ester (5,6-Cl2-IAA-Me) at different concentrations, respectively. Each chemical produced the maximum number of adventitious roots at a different concentration. Compared with IBA treatment, 5,6-Cl2-IAA-Me and BSAA treatments significantly increased root numbers on hypocotyl cuttings at lower concentration, particularly of 5,6-Cl2-IAA-Me treatment. Combinations of paclobutrazol (PB) with either 5,6-Cl2-IAA-Me or BSAA significantly stimulated the production of more adventitious roots than either chemical alone or combined. Capillary electrophoresis analysis have shown that the levels of IAA, IBA and BSAA in IBA plus PB or BSAA plus PB treatments were higher than those of IBA or BSAA alone. It was suggested that the cause of the synergistic effect of IBA (or BSAA) plus PB treatment might be due to increased endogenous auxin level. The activities of peroxidase and IAA oxidase in the rooting zone coincided with root development, indicating that the activities of these two enzymes were positively correlated to rooting. Peroxidase and IAA oxidase activity in all treatments started 24 h and 12 h after cutting, respectively. It is suggested that the major role of IAA oxidase differed from that of peroxidase in adventitious root formation.     

Metabolic changes during rooting in stem cuttings of Casuarina equisetifolia L.: effects of auxin, the sex and the type of cutting on rooting

Rooting in terminal shoot and lateral shoot cuttings from 10-year-old elite trees of Casuarina equisetifolia L. in different sex groups was achieved after 20 days when the basal ends of the cuttings were dipped for 3 h in 20 ppm indole-3-butyric acid (IBA). Shoots derived from male plants rooted better than their female and monoecious counterparts, and the lateral shoots were more responsive to rooting than the terminal shoots. During rooting, the metabolic activities varied in both lateral shoot and terminal shoot cuttings derived from plants under different sex groups. Peroxidase and polyphenoloxidase activities were high during root initiation and showed a sharp decline thereafter. The polyphenoloxidase activity was higher in the lateral shoot than the terminal shoot cuttings. The rooted plantlets survived and established well in the field.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone     

Evaluation of antioxidant bioindicators and growth responses in <Emphasis Type="Italic">Malva parviflora</Emphasis> L. exposed to cadmium

In this study, the effect of cadmium (Cd) uptake and concentration on some growth and biochemical responses were investigated in Malva parviflora under Cd treatments including 0, 10, 50 and 100 µM. The shoots and roots were able to accumulate Cd. However, increased Cd dose led to a considerable Cd content in the roots. Cd stress decreased growth, increased lipid peroxidation and also enhanced proline and ascorbic acid contents in both shoots and roots. Chlorophyll and carotenoid contents decreased in the plants with the increasing Cd concentration. While the activities of catalase (CAT) and superoxide dismutase (SOD) increased in the shoots under different Cd doses, these activities decreased in the roots as compared to the control. Both shoots and roots demonstrated a significant increase in guaiacol peroxidase activity in response to Cd stress. Contrary to the aboveground parts, the roots subjected to Cd doses showed a rise in protein content. Despite higher Cd content in the roots, it seems that CAT and SOD do not play a key role in detoxification of Cd-induced oxidative stress. These findings confirm that reduced biomass and growth under Cd stress can be due to an increase in oxidative stress and a decrease in photosynthetic pigment content. The present study clearly indicates that the shoots and roots exploit different tolerance behaviors to alleviate Cd-induced oxidative stress in M. parviflora.     

Promotion of adventitious root formation by 4-chlororesorcinol: A polyphenol oxidase inhibitor

The extent of rooting in cuttings of Phaseolus vulgaris L., and Vigna radiata Wilcz. was affected by 4-chlororesorcinol, a polyphenol oxidase inhibitor. More root primordia and more roots were formed after 4-chlororesorcinol treatment both with and without 10^-5M Indole butyric acid. Promotion of rooting was observed also in cuttings of Elaeagnus pungens, Gypsophilia elegans and Kalanchoe blossfeldiana. The enhancement in bean and mung bean was accompanied by a concomitant wider spatial distribution of the primordia and the resulting adventitious roots. The formation of primordia in the treated cuttings was delayed by 12–24 hours, compared to untreated cuttings. The treatment was effective only when given during the first hours after the preparation of the cutting of bean and mung bean, suggesting involvement in the initiation stage. Hypocotyl extracts of mung bean cuttings, pretreated with 4-chlororesorcinol, exhibited reduced polyphenol oxidase activity. The inhibition was not reversed by washing of the treated extract in 50% acetone or by an overnight dialysis, suggesting tight or maybe even irreversible binding of the inhibitor to the enzyme.Abbreviations 4-CR 4-chlororesorcinol - IBA Indole butyric acid - PPO polyphenol oxidase     

Structure and development of adventitious roots in Salix caprea and S. caprea × S. viminalis

Formation and structure of adventitious roots in cuttings of two clones of S. caprea and a hybrid betwen S. caprea and 5. viminalis was studied with light microscopy (LM) and transmission electron microscopy (TEM). The hybrid contained large preformed root primordia, which in cuttings placed in water developed into roots in only three days. One of the S. caprea clones contained minute preformed root primordia, which developed into roots in about eight days. Treatment with indolebutyric acid (IBA) increased the percentage of rooted cuttings and the number of roots formed. Roots emerging from IBA-treated cuttings contained both mature protophloem and protoxylem, while in roots of untreated cuttings only some sieve elements of the protophloem were mature. In the other 5. caprea clone no preformed root primordia were detected, but after treatment with IBA roots appeared in about two weeks. The cambium in treated stems produced a large number of cells, most of which differentiated into xylem. Root primordia were initiated in the newly produced tissue external to the cambium. The roots contained both mature protophloem and protoxylem at emergence. A few roots emerged also from extensive callus tissues formed external to the basal end of the cuttings. Cell enlargement and cell divisions in various parts of the base of the cuttings caused disruption of the peripheral tissues, which made the cuttings susceptible to infection by microorganisms.     

NAA处理桉树插条后IAAO活性与生根的关系

尾叶桉MLA无性系(简称MLA)为难生根植物,尾叶桉U₆无性系(简称U₆)和刚果12号桉W₅无性系(简称W₅)为相对易生根植物。MLA插条内的吲哚乙酸氧化酶(IAAO)活性较U₆、W₅的高。用萘乙酸(NAA)处理桉树插条后,在扦插生根的不同阶段,插条内IAAO活性呈现规律性变化;可溶性蛋白质含量呈上升趋势。本文讨论了IAAO与桉树插条生根的关系。     

Polyamine catabolism influences antioxidative defense mechanism in shoots and roots of five wheat genotypes under high temperature stress

Effect of high temperature stress on polyamine catabolism and antioxidant enzyme activity in relation to glutathione, ascorbate and proline accumulation was studied in five wheat (Triticum aestivum L.) genotypes (differently susceptible to temperature stress). High temperature significantly increased the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and glutathione S-transferase (GST) in shoots of all genotypes. Higher activities of GPX in C 306, C 273 and APX in PBW 550, PBW 343 and PBW 534 demonstrate their important role in scavenging H₂O₂. Conversely, high temperature stress led to a significant decline in SOD, CAT, APX and GPX activities of roots with a subsequent increase in diamine oxidase (DAO) and polyamine oxidase (PAO) activities especially in PBW 550 and PBW 343. The concentration of ascorbic acid declined with the imposition of heat stress, however, polyamines responded to high temperature stress by increasing spermidine and spermine levels and decreasing putrescine levels. After exposure to high temperature, proline accumulation was significantly decreased in roots and increased in shoots though maximum concentration was achieved in C 306 genotype. Apparently, the wheat seedlings respond to high temperature mediated increase in reactive oxygen species (ROS) production by altering antioxidative defense mechanism and polyamine catabolism though differentially in five wheat genotypes. Among five genotypes studied, C 306 and C 273 seem to be better protected against temperature stress. The results suggested that shoots were more resistant against the destructive effects of ROS as is indicated by low levels of thiobarbituric acid reactive substances under high temperature stress.     

EXPERIMENTS WITH ROOT CUTTINGS OF BRUSSELS SPROUT

A technique for the propagation of Brussels sprout by means of root cuttings is described. Adventitious shoots arise exogenously on callus tissue which develops around the base of side roots. Cuttings sometimes rot without forming adventitious shoots, and cuttings which remain sound do not all produce shoots. Rotting may largely be prevented by planting cuttings with the proximal end exposed above the surface of the medium, and by allowing the root portions to dry before planting. Surface sterilization with mercuric chloride controls rotting but reduces bud formation. Individual plants differ in their capacity to form buds on root cuttings, and this difference is carried by the clones derived from them. Portions of root form more buds if cut into several pieces than if planted intact.     

沙生柽柳扦插生根过程插穗相关理化特征分析

选取沙生柽柳半木质化枝条进行苗床扦插,通过实验测定插穗生根过程中内源激素(IAA、GA3、ZR、ABA)含量、可溶性营养物质(糖、蛋白质)含量及相关氧化酶(PPO、POD、SOD、IAAO)活性的动态变化特征,探讨沙生柽柳插穗扦插生根机理。结果表明:(1)沙生柽柳插穗内源激素含量随生根进程而发生变化,其中,IAA含量在扦插35d最大,并出现较大的波动变化;ZR含量在扦插55d前后变化明显,呈现低水平向高水平转化趋势;ABA、GA3含量依次呈先升高后降低再升高的变化过程,并在扦插15d和55d(80d)呈现变化的峰值和谷值。(2)沙生柽柳扦插生根与相关氧化酶活性密切相关,其中,POD、IAAO活性在插穗扦插35d后长时间保持较高水平,直至插穗生根后POD活性明显降低,IAAO活性有所增加;PPO、SOD活性则在插穗扦插15d保持较高活性,且PPO活性的变化均匀,SOD活性的高低交替变化明显。(3)在沙生柽柳扦插生根期间,插穗可溶性糖含量呈现生根前消耗减少与生根后积累增加两大变化过程,可溶性蛋白质含量表现为扦插后逐步积累增加的变化趋势。研究表明,高水平的IAA、ZR和低水平的GA3、ABA共同调控着沙生柽柳插穗生根;IAA能够通过促进插穗POD、PPO、IAAO活性变化来影响生根,较高的POD、IAAO活性可调节插穗IAA水平,高水平的PPO活性则催化插穗IAA-酚酸复合物的形成,进而诱导插穗生根。