Chemical induction of adventitious root formation in Taxus baccata cuttings

The effect of some auxins (IBA and NAA), phenolic compounds (phloroglucinol, gentisic acid and coumarin), a combination of auxins and phenolics, and a systemic fungicide (Bavistin) have been examined for stimulatory effects on adventitious root formation in stem cuttings (current season's growth) of Taxus baccata L. In general lower concentration (0.25 mM) of both IBA and NAA was more effective in inducing rooting of cuttings taken from both male and female trees. The combined treatment of IBA+NAA (0.25 mM each) showed some success in cuttings from male trees only (55%, compared to 15% rooting in cuttings from female trees). Generally, the callus formation was quite high (70%) in all auxin treatments (alone or in combination). Among the phenolics, 40% rooting success was achieved with phloroglucinol only, while coumarin and gentisic acid were ineffective. The combined treatment of auxins and phenolics also failed to promote rooting. On the other hand, Bavistin was extremely effective for callusing (90%) as well as rooting (80%). The effectiveness of various compounds tested for rooting of young stem cuttings declined in the order: 0.25 mM IBA>0.05% Bavistin>0.25 mM NAA>1.25 mM IBA>15 mM phloroglucinol>IBA+NAA (0.25 mM each). In addition to the auxins, IBA and NAA that are widely used for commercial propagation, the auxin-like properties of the fungicide Bavistin could be exploited for adventitious rooting in T. baccata, and in other plant species.     

Aeroponics for adventitious rhizogenesis in evergreen haloxeric tree <Emphasis Type="Italic">Tamarix aphylla</Emphasis> (L.) Karst.: influence of exogenous auxins and cutting type

Tamarix aphylla (L.) Karst., a drought resistant halophyte tree, is an agroforestry species which can be used for reclamation of waterlogged saline and marginal lands. Due to very low seed viability and unsuitable conditions for seed germination, the tree is becoming rare in Indian Thar desert. Present study concerns the evaluation of aeroponics technique for vegetative propagation of T. aphylla. Effect of various exogenous auxins (indole-3-acetic acid, indole-3-butyric acid, naphthalene acetic acid) at different concentrations (0.0, 1.0, 2.0, 3.0, 5.0, 10.0 mg l^?1) was examined for induction of adventitious rooting and other morphological features. Among all three auxins tested individually, maximum rooting response (79%) was observed with IBA 2.0 mg l^?1. However, stem cuttings treated with a combination of auxins (2.0 mg l^?1 IBA and 1.0 mg l^?1 IAA) for 15 min resulted in 87% of rooting response. Among three types of stem cuttings (apical shoot, newly sprouted cuttings, mature stem cuttings), maximum rooting (~ 90%) was observed on mature stem cuttings. Number of roots and root length were significantly higher in aeroponically rooted stem cuttings as compared to stem cuttings rooted in soil conditions. Successfully rooted and sprouted plants were transferred to polybags with 95% survival rate. This is the first report on aeroponic culture of Tamarix aphylla which can be utilized in agroforestry practices, marginal land reclamation and physiological studies.     

The effect of ammonium ions on uptake of glutamine and other amino compounds by cultured cells of rapeseed

Rooting responses and ethylene production by hypocotyl cuttings from etiolated mung-bean seedlings treated with the auxins -naphthaleneacetic acid, -(indole-3)-n-butyric acid (IBA) and 2,4,5-trichloro-phenoxypropionic acid were determined. There was no relationship between the abilities of the auxins to induce root formation and their capacities for inducing ethylene production. Studies with mixtures of 3-indoleacetic acid, a poor stimulator of rooting but an effective inducer of ethylene production, and IBA, an effective rooting stimulator but a poor inducer of ethylene production, exposure of cuttings to ethylene or (2-chloroethyl) phosphonic acid (Ethephon), hypobaric storage (150 mb) of treated cuttings, and exposure of auxin-treated cuttings to 7% CO₂ also indicated that ethylene is not directly involved in initiation of adventitious roots in this plant material.Abbreviations IAA indole-3-acetic acid - IBA -(indole-3)-n-butyric acid - NAA -naphthaleneacetic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid     

Vegetative propagation of three mangrove tree species by cuttings and air layering

Large scale propagation of three mangrove species, Excoecaria agallocha, Heritiera fomesand Intsia bijugausing cuttings and air layering was attempted. The effect of auxins and season on rooting potential of these mangrove species was recorded. Maximum rooting was recorded when the cuttings and air layers were treated with IBA alone up to 2500ppm in all the three species. October was found to be best followed by January for the plantation of cuttings and initiation of air layers. All the plants were hardened and field transferred into the mangrove forests of Pichavaram, Tamilnadu, India.     

Effect of auxins on adventitious rooting from stem cuttings of candidate plus tree Pongamia pinnata (L.), a potential biodiesel plant

Pongamia pinnata, commercially important tree species used to produce biofuels, is known for its multipurpose benefits and its role in agro-forestry. Present study examines the amenability of vegetative propagation and effect of maturation in candidate plus tree P. pinnata through rooting of stem cuttings treated with varying concentrations and combinations of auxins. The performance of the cuttings was evaluated using SAS GLM software and the data were analyzed as a one-way classified data with and without sub sampling for inferring auxin concentration that can be included in programmes aimed at genetic improvement of the tree species. All auxin treatments promoted sprouting and at lower concentrations triggered/enhanced rooting of cuttings. The effectiveness was in the order of IBA > NAA > IAA when applied singly. IBA at 4.92 mM was found to be most effective where rooting percentage and number of roots were significantly higher (P < 0.01) than in control. However higher concentrations of auxins above 7 mM in general inhibited the rooting of cuttings. The interaction among auxins was found to be effective in root induction and differentiation and the most stimulating effects were observed in three-component mixture. The effect of other cutting characteristics such as juvenility and cutting position on rooting is also discussed.     

Metabolic changes during rooting in stem cuttings of five mangrove species

Vegetative propagation through rooting in stem cuttings in five tree mangroves namely Bruguiera parviflora, Cynometra iripa, Excoecaria agallocha, Heritiera fomes, and Thespesia populnea using IAA, IBA and NAA was reported. Spectacular increase in the root number was noted in the cuttings of H. fomes and C. iripa treated together with IBA (5000 ppm) and NAA (2500 ppm). The highest number of roots was obtained with IBA (2500 ppm) and NAA (500 ppm) in E. agallocha. B. parviflora and T. populnea responded better to IAA and IBA treatment. The species specific variation in the rooting response to exogenous application of auxins was reflected in the metabolic changes during initiation and development of roots in cuttings. Biochemical analysis showed increase of reducing sugar in the above-girdled tissues at initiation as well as subsequent development of roots which was further enhanced by the use of auxins. Decreases in the total sugar, total carbohydrate and polyphenols and increase in total nitrogen were recorded in the girdled tissues and the high C/N ratio at the initial stage helped in initiation of roots in all the species. Interaction of IBA and NAA promoted starch hydrolysis better than IAA and IBA during root development and subsequently reduced the C/N ratio and increased the protein-nitrogen activity during root development which suggest the auxin influenced mobilization of nitrogen to the rooting zone.Abbreviations IAA Indole-3-acetic acid - IBA Indole-butyric acid - NAA A-naphthalene acetic acid     

Effect of Auxins and Light on Rooting Stem Cuttings of Populus nigra Salix tetrasperma, Ipomea fistulosa and Hibiscus notodus in Relation to Polarity

The apical and basal ends of stem cuttings of Populus nigra, Salix tetrasperma, Ipomoea fistulosa and Hibiscus notodus were treated with 10 mg/l solutions of IAA and IBA for 24 hours and were planted either erect or inverted both in light and dark. Observations for the number of cuttings that rooted and the roots produced on them were recorded at weekly intervals. In Salix, Ipomoea and Hibiscus rooting was more on cuttings planted erect, while in populus it did not differ much with the manner of planting. The reduced rooting in inverted cuttings may be ascribed to the low level of endogenous auxin at the apex due to polar transport. An exogenous application of auxins enhanced rooting on inverted cuttings. In dark, roots on Populus and Salix cuttings were produced both above and within the rooting medium. The weak polarity of these two plants may be due to the potential root primordia reported in their stem. The formation of callus occurred on the top of Populus cuttings whether planted erect or inverted but it differentiated into branches on erect cuttings only. In those planted in an inverted position the callus failed to differentiate in spite of the application of kinetin, auxins, TIBA, coumarin and sucrose, and dried ultimately.     

Effect of Non-auxin Chemicals on Translocation of Auxins in Cuttings of Phaseolus vulgaris (L.)

Indole, -naphthol, pyrogallol, coumarin, and salicylic acidinteracted with the auxins, IAA (indol-3yl-acetic acid), NAA(naphth-lyl-acetic acid), and 2, 4-D (2, 4-dichlorophenoxyaceticacid), supplied to the basal ends of cuttings of Phaseolus vulgaris(L.), giving synergistic or antagonistic effects in root formation.Antagonism in rooting was always associated with increased accumulationof radiocarbon from carboxyl-¹⁴C-labelled auxins in the topsof the cuttings. Distribution of auxin over a greater lengthof the cutting was accompanied by a reduction in root formation.The chemicals which synergized auxin-induced root formationdid not promote accumulation of radiocarbon of the exogenouslyapplied labelled auxins in the upper parts of the cuttings.     

A simple and efficient method for clonal propagation of Casuarina sumatrana (de Vriese) L. Johnson

A new and efficient method for clonal propagation of Casuarina sumatrana by rooting stem cuttings is described. High percentage (about 60–70%) of rooting was achieved with mature softwood stem cuttings. A quick-dip of 5 s in NAA (1–10mM) solution followed by sand culture under high humidity were required for a high rate of survival and rooting of stem cuttings. A simple, closed chamber propagation system, using fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film), was successfully developed for the rooting of stem cuttings without mist. Rooted cuttings inoculated with Frankia were easily transplanted and established in field conditions with very low (about 3%) mortality. The significance of these findings for mass clonal propagation of C. sumatrana is discussed.Abbreviations IBA indole-3-butyric acid - NAA -naphthaleneacetic acid     

Seasonal Changes in Auxin Effects on Rooting of Stem Cuttings of Populus nigra and its Relationship with Mobilization of Starch

Stem cuttings of Populus nigra were treated with 10 and 100 mg/1 each of IAA., IBA, 2,4-D and NAA at one month intervals and observations were recorded for the morphophysiological status of the branches, their starch content and their rooting response. — The first phase characterized by delayed, short and scarce roots and the high starch content of cuttings coincided with the onset of winter dormancy in November lasting through February. It was followed by a phase of vigorous rooting and low starch content of cuttings coinciding with the renovation of growth activity in February lasting through October, except in April and May when rooting was more or less completely nullified. — The poor rooting in winter was caused by low activity of hydrolyzing enzymes not mobilizing starch into soluble sugars; and profuse rooting during active growth period by high activity of hydrolyzing enzymes caused by endogenous auxin, resulting in mobilization of reserved food materials necessary for the initiation and development of roots. The low rooting in April and May is ascribed to the fact that bulk of the mobilized food was used up in the growth of sprouted branches leaving very little for rooting when these cuttings were planted. — The seasonal changes in the effectiveness of exogenously applied auxins also appear to be related with the level of endogenous auxin. In June endogenous auxin was high due to high meristematic activity, the exogenously applied auxins raising it to supra-optimal levels that were inhibitory. On the other hand, in October exogenously applied auxins enhanced rooting by raising it to an optimal level as the production of endogenous auxin had been decreasing gradually due to lowering temperatures. — The results demonstrate that auxin effect on differential rooting with season in this plant is determined by the physio-morphological status of the branches that govern the production of endogenous auxin and is mediated primarily through its effect on mobilization of reserve food materials caused by enhanced activity of hydrolytic enzymes.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

Evidence for the presence of a native,non-auxinic rooting promoter in avocado (Persea americana Mill.)

The presence of a rooting promoter in paratially purified extracts of avocado (Persea amricana Mill.) organs has been demonstrated using the mung bean rooting bioassay. Extraction with 80% methanol was followed by partition into diethyl ether, paper chromatography (PC) and 3 steps of thin layer chromatography (TLC). The number of roots induced by the rooting promoter in the absence of exogenous auxin was 5 to 7 times higher than that of the water control and 50% higher than by 4-(indol-3-yl) butyric acid (IBA) at its reported optimum concentration. Rooting of tomato, Coleus and young avocado cuttings was also enhanced by the rooting promoter. The rooting promoter was inhibitory in the wheat coleoptile section elongation bioassay for auxins and had slight inhibitory activity in the split pea stem curvature test.The biological properties of the avocado rooting promoter may be comparable to those of -(p-chlorophenoxy) isobutyric acid (PCIB) which acts as an anti-auxin in certain bioassays and, nevertheless, promotes the rooting of mung bean cuttings.     

Growth Performance of Cuttings Raised from in vitro and in vivo Propagated Stock Plants of Rosa damascena Mill.

Comparative studies on rooting and growth performance of cuttings raised from in vitro and in vivo grown plants of Rosa damascena are described. Cuttings were treated with different auxins and upon transfer to soil their growth performance was recorded. Overall, the auxin treated cuttings of in vitro raised plants responded better than the cuttings of in vivo raised plants. Optimal response for percentage of rooting, root number, root length and bottom breaks was observed at 100 mg dm^–3 IBA. The cuttings derived from in vitro raised plants showed a significantly better response for percent rooting, root number, root length and bottom buds in control treatments.     

Ethylene and auxin-ethylene interaction in adventitious root formation in mung bean (Vigna radiata) cuttings

The role of ethylene in adventitious root formation and its involvement in auxin-induced rooting were investigated in cuttings ofVigna radiata (L.). Treatment with 30 M indole-3-acetic acid (IAA) for 24 h slightly inhibited rooting, whereas the same concentration of indole-3-butyric acid (IBA) significantly stimulated it. Ethylene derived from 1-aminocyclopropane-1-carboxylic acid (ACC) increased the number of adventitious roots but inhibited their emergence and elongation. Endogenous levels of ethylene, ACC, and malonyl-ACC (MACC) were initially higher in cuttings treated with IAA. This trend was quickly reversed, and cuttings, particularly hypocotyls, treated with IBA produced higher levels of ethylene and had more ACC and MACC during most of the rooting process. Aminoethoxyvinylglycine significantly inhibited rooting, but its inhibitory effect could not be reversed by ACC. The data suggest that the stimulating effect of IBA on rooting is closely associated with its induction of ACC and ethylene biosynthesis.     

Fluctuations of different endogenous phenolic compounds and cinnamic acid in the first days of the rooting process of cherry rootstock 'GiSelA 5' leafy cuttings

The relationship between the phenol composition of rooting zones and rootability was studied in the first days after the establishment of cuttings. The trial included two different types of cuttings (basal and terminal). Additionally, the influence of exogenously applied auxin (IBA) was observed. The best rooting results (55.6%) were achieved with terminal IBA treated cuttings, while only 1.9% of basal cuttings formed roots. The auxin treatment increased the root formation in terminal, but not in basal cuttings. Low rooting rate of basal cuttings was probably due to higher lignification rate of the basal tissue which can represent a mechanical barrier for root emergence. When measuring phenolic compounds and cinnamic acid, terminal cuttings contained higher (rutin, vanillic acid, (-)-epicatechin, caffeic acid and sinapinic acid) or equal concentrations of detected phenols as basal cuttings, while applied auxin did not influence the level of any of discussed phenolics, neither of cinnamic acid. It is to assume that cuttings for starting of root induction phase should contain certain levels of several phenolic compounds, but higher influence on rooting success is to be ascribed to the impact of the auxin level. During the time of the experiment concentrations of monophenols sinapinic acid and vanillic acid rapidly decreased. This decrease was more pronounced in terminal cuttings, which might have a better mechanism of lowering those two compounds to which a negative influence on rooting is ascribed. Fluctuations and differences between treatments of other phenolics were not significant enough to influence the rooting process.     

Stock plant etiolation and stem banding effect on the auxin dose-response of rooting in stem cuttings of Carpinus betulus L. ‘Fastigiata’

Experiments were undertaken to determine the effect of stock plant etiolation and stem banding, prior to cutting propagation, on the auxin dose-response of rooting in Carpinus betulus L. Fastigiata stem cuttings. Stock plants were forced in a greenhouse, etiolated for 10 days and banded with black, light-tight Velcro for 8 weeks. Indole-3-butyric acid was applied to cuttings at concentrations ranging from 0 to 79 mM. Rooting percentages and numbers increased to a peak reponse at 20 mM in light-grown and 40 mM in etiolated shoots, followed by an inhibition at higher concentrations for all except etiolated and banded shoots. Cuttings prepared from shoots which had been etiolated or banded rooted better than controls at low and optimal IBA concentrations. Cuttings from shoots receiving both etiolation and banding also yielded higher rooting percentages and more roots per rooted cutting. Furthermore, etiolation and banding reduced the sensitivity of cuttings to supra-optimal auxin-induced inhibition of adventitious root initiation.     

N-phenyl indolyl-3-biityraitiide and phenyl indole-3-thiolobutyrate enhance adventitious root primordium development

N-phenyl indoryl-3-butyramide (NP-IBA) and phenyl indole-3-thiolobutyrate (P-ITB) are new synthetic auxins that enhance rooting of bean ( Phaseolus vulgaris cv. Top Crop) hypocotyl cuttings and jack pine ( Pinus banksiana Lamb.) seedling cuttings. In comparison with an equal concentration of indole-3-butyric acid (IBA), NP-IBA treatment resulted in greater rooting of bean and jack pine seedling cuttings. IBA and P-ITB treatment at equal concentration were equally effective in inducing rooting of bean cuttings. However, in comparison with an equal concentration of IBA, P-ITB treatment promoted greater rooting of jack pine seedling cuttings, but P-ITB treatment was less effective than NP-IBA treatment. Application of crystalline chemical to jack pine seedling cuttings indicated lesser toxic effects and earlier and greater root-promoting effects of P-ITB and NP-IBA treatments, in comparison with IBA treatment. The earlier rooting of NP-IBA-treated jack pine seedling cuttings was positively related to increase in fresh weight of basal 1 cm stem before primordia were initiated. Weight of basal steins of NP-IBA-treated cuttings initially increased during propagation at day 2 in comparison with the control, and at day 3 in comparison with IBA-treated cuttings. Results that were obtained with seedling jack pine cuttings were generally confirmed for cuttings from 7-year-old trees.     

Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport

Indole-3-butyric acid (IBA) was recently identified by GC/MS analysis as an endogenous constituent of various plants. Plant tissues contained 9 ng g^?1 fresh weight of free IBA and 37 ng g^?1 fresh weight of total IBA, compared to 26 ng g^?1 and 52 ng g^?1 fresh weight of free and total indole-3-acetic acid (IAA), respectively. IBA level was found to increase during plant development, but never reached the level of IAA. It is generally assumed that the greater ability of IBA as compared with IAA to promote rooting is due to its relatively higher stability. Indeed, the concentrations of IAA and IBA in autoclaved medium were reduced by 40% and 20%, respectively, compared with filter sterilized controls. In liquid medium, IAA was more sensitive than IBA to non-biological degradation. However, in all plant tissues tested, both auxins were found to be metabolized rapidly and conjugated at the same rate with amino acids or sugar. Studies of auxin transport showed that IAA was transported faster than IBA. The velocities of some of the auxins tested were 7. 5 mm h^?1 for IAA, 6. 7 mm h^?1 for naphthaleneacetic acid (NAA) and only 3. 2 mm h^?1 for IBA. Like IAA, IBA was transported predominantly in a basipetal direction (polar transport). After application of ³H-IBA to cuttings of various plants, most of the label remained in the bases of the cuttings. Easy-to-root cultivars were found to absorb more of the auxin and transport more of it to the leaves. It has been postulated that easy-to-root, as opposed to the difficult-to-root cultivars, have the ability to hydrolyze auxin conjugates at the appropriate time to release free auxin which may promote root initiation. This theory is supported by reports on increased levels of free auxin in the bases of cuttings prior to rooting. The auxin conjugate probably acts as a ‘slow-release’ hormone in the tissues. Easy-to-root cultivars were also able to convert IBA to IAA which accumulated in the cutting bases prior to rooting. IAA conjugates, but not IBA conjugates, were subject to oxidation, and thus deactivation. The efficiency of the two auxins in root induction therefore seems to depend on the stability of their conjugates. The higher rooting promotion of IBA was also ascribed to the fact that its level remained elevated longer than that of IAA, even though IBA was metabolized in the tissue. IAA was converted to IBA by seedlings of corn and Arabidopsis. The K_m value for IBA formation was low (approximately 20 μM), indicating high affinity for the substrate. That means that small amounts of IAA (only a fraction of the total IAA in the plant tissues) can be converted to IBA. It was suggested that IBA is formed by the acetylation of IAA with acetyl-CoA in the carboxyl position via a biosynthetic pathway analogous to the primary steps of fatty acid biosynthesis, where acetyl moieties are transferred to an acceptor molecule. Incubation of the soluble enzyme fraction from Arabidopsis with ³H-IBA, IBA and UDP-glucose resulted in a product that was identified tentatively as IBA glucose (IBGIc). IBGIc was detected only during the first 30 min of incubation, showing that it might be converted rapidly to another conjugate.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Morpho-anatomical and biochemical changes associated with rooting of micropropagated ninebark cuttings

Ninebark (Physocarpus opulifolius) is an attractive ornamental shrub with poor rooting characteristics in some cultivars, which is a limiting factor in commercial production This study was designed to optimize rooting conditions of ninebark cuttings and to observe the effect of exogenous auxin IBA on some morpho-anatomical and biochemical changes associated with rhizogenesis in the in vitro conditions. Both auxins under study: the indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) gave comparable effects but the combination of ½ MS?+?1 mg·L^?1 IBA was the most cost effective for all rooting parameters. Anatomical changes at the cuttings’ bases during root formation were typical for woody plants and they were accelerated by auxin in the culture medium. High levels of the endogenous indole acid and hydrogen peroxide were temporarily associated with intensive cell divisions in cuttings, and the polyphenolic acid contents kept increasing during rooting above the initial levels and those in controls.