Subsequent Root Growth Research Articles

Photolysis of caged cytokinin in single cells of Arabidopsis thaliana

BackgroundCytokinins are a class of phytohormone that play a crucial role in the development of plants. They are involved in the regulation of nearly every aspect of plant growth, from germination to senescence. The role of cytokinins in many developmental programs is complex and varies both spatially and temporally. Current techniques used to investigate the functions of cytokinins in plant development lack this spatial and temporal resolution required to observe cell-type specific effects.ResultsTo this end, we present a method of activating a caged cytokinin in single cells. A caged benzyladenine was synthesized, along with caged adenine as a negative control. In vitro testing confirmed ultraviolet light-mediated uncaging, and subsequent root growth assays demonstrated that uncaging produced a cytokinin phenotype. This uncaging was confined to single cells using multiphoton confocal microscopy. Using an Arabidopsis thaliana cytokinin reporter line expressing TCSn::GFP, the resulting GFP expression was confined to the uncaging region, including in single cells. This study presents a novel cell-targeted method of cytokinin delivery, which has the potential to elucidate a broad range of processes in plant development.ConclusionsWe combined multiphoton confocal microscopy and a caged cytokinin treatment, allowing cell type-specific uncaging of a cytokinin in Arabidopsis roots.

X‐ray CT reveals 4D root system development and lateral root responses to nitrate in soil

AbstractThe spatial arrangement of the root system, termed root system architecture, is important for resource acquisition as it directly affects the soil zone explored. Methods for phenotyping roots are mostly destructive, which prevents analysis of roots over time as they grow. Here, we used X‐ray microcomputed tomography (μCT) to non‐invasively characterize wheat (Triticum aestivum L.) seedling root development across time under high and low nitrate nutrition. Roots were imaged multiple times with the 3D models co‐aligned and timestamped in the architectural plant model OpenSimRoot for subsequent root growth and nitrate uptake simulations. Through 4D imaging, we found that lateral root traits were highly responsive to nitrate limitation in soil with greater lateral root length under low N. The root growth model using all μCT root scans was comparable to a parameterized model using only the final root scan in the series. In a second μCT experiment, root growth and nitrate uptake simulations of candidate wheat genotypes found significant root growth and uptake differences between lines. A high nitrate uptake wheat line selected from field data had a greater lateral root count and length at seedling growth stage compared with a low uptake line.

Characterizing the Utility of the Root-to-Shoot Ratio in Douglas-Fir Seedling Production

Nursery-grown tree seedlings are a vital component of successful restoration and reforestation programs, useful when calls for increased planting for industrial forest management are made, and a tool for climate change mitigation. One of the most extensively planted and studied trees in Western North America is Douglas-fir. Building on that body of work, this review was conducted to identify if the root-to-shoot ratio (root:shoot, R:S), a commonly referred-to metric in reforestation planning, yields meaningful guidance for producing seedlings that are better able to establish across a variety of field conditions. The results indicated that there is wide variability in R:S of nursery-grown seedlings. The relationship between R:S and subsequent root growth and seedling survival varies depending on Douglas-fir variety, seedling stocktypes, and site conditions. The biological and physiological basis for using R:S remains, and likely could be used to enhance seedling quality; however, there is an ongoing need for planning and collaboration between researchers and practitioners to identify how to best deploy this evaluation tool.

Impact of Superabsorbent Polymers and Variety on Yield, Quality and Physiological Parameters of the Sugar Beet (Beta vulgaris prov. Altissima Doell).

In this study, we focus on the mitigation of the negative impact of drought using the application of superabsorbent polymers (SAPs) to seed. One way to monitor drought and quantify its impact on crops in field conditions is the nondestructive measurement of physiological processes of the crops using spectral indexes LAI and PRI during vegetation. Therefore, during 2018 and 2019, the increase in biomass and intensity of photosynthetic activity was monitored, and the effect of the SAPs application on the yield parameters of the sugar beet was evaluated in the trial conditions (control, SAPs) at the end of the vegetation period. Through statistical analysis, the significant impact (α ≤ 0.01) of SAPs application on the values of spectral indexes LAI and PRI as well as root and white sugar yields was found. Although the sugar content difference between SAPs and control conditions was not statistically significant, SAPs had a positive influence on the value of this parameter. It was found through periodic monitoring of spectral indexes during the growing period that the crop in the SAPs condition showed higher values of PRI at the beginning of vegetation, which was caused by the accumulation of moisture in the vicinity of the seed and subsequent faster growth of roots and photosynthetic apparatus. Moreover, the values of LAI were significantly higher (α ≤ 0.01) in the SAPs condition throughout the vegetation period. In the interaction evaluation, we confirmed that in both years the values of LAI were higher in the condition with SAPs compared with the control. In contrast, the PRI values were significantly different across conditions. The interaction of conditions with variety showed that the variety Brian obtained higher values of LAI and PRI in the SAPs condition. The correlation analysis found a positive correlation between spectral indexes LAI:PRI (r = 0.6184**), and between LAI:RY (r = 0.6715**), LAI:WSY (r = 0.5760**), and PRI:RY (r = 0.5038*), which confirms the close relationship between physiological processes in the plant and the size of its yield.

Subsoil compaction effect on spatio-temporal root growth, reuse of biopores and crop yield of spring barley

Subsoil compaction in arable farming caused by increasingly heavy machinery results in increased subsoil strength and reduced aeration and water flow. This can restrict root growth and reduce crop yield. To mitigate these effects of subsoil compaction, we included a deep-rooted catch crop of fodder radish (Raphanus sativus var. oleifera) in a rotation of spring barley (Hordeum vulgare). The objective was to determine the effect of subsoil compaction on root growth and grain yield of barley, and to investigate the creation and use of biopores for subsequent root growth. Studies were carried out in 2013–2016 in a soil compaction experiment established in 2010 on a sandy loam. The soil had for four consecutive years (2010–2013) been compacted with multi-passes in an 8 Mg wheel load treatment (M8) or had not been compacted (Control). Spring barley was grown every year and fodder radish was grown as a catch crop from harvest to winter in 2013–2016. Penetration resistance to 90 cm depth was measured in 2015 at a soil water content near field capacity. Root growth and biopore reuse were investigated using horizontal minirhizotrons installed at 30, 50 and 70 cm depth from 2013–2016. Spring barley grain yield was recorded annually. We found that M8 caused critical subsoil compaction with penetration resistance peaking at 3.5 MPa in the upper subsoil (30−40 cm depth). Subsoil compaction reduced root growth by c. 50 % at 30 cm depth and barley yields by 6–8 % compared to the Control in three years after cessation of the compaction treatment. The number of biopores that were reused were higher for fodder radish than for spring barley, 4.1 % and 3.1 %, respectively, but did not differ between compaction treatments. Our study displayed a marked, negative residual effect of soil compaction on above- and belowground crop growth. There was no tendency towards a recovery within the years studied (1–3 years after compaction). Severe subsoil compaction is a widespread problem and therefore we call for more studies on the spatio-temporal dynamics of the creation and subsequent use of biopores for root growth as these are essential for the recovery of compacted subsoils.

Nitrogen loading of Eucalyptus globulus seedlings: nutritional dynamics and influence on morphology and root growth potential

Achieving successful outplanting of trees on increasingly harsher sites is a global concern. In Chile, for example, new Eucalyptus globulus plantations are being targeted to poorer, low fertility sites where additional stresses often negatively affect seedling performance during the first field season. Nitrogen-loading during the later stages of nursery production can, however, significantly improve seedling physiological performance on harsher sites by increasing plant growth and nutrient storage reserves for subsequent root growth after outplanting. Maximizing these benefits, and applying the concept of loading throughout the entire crop cycle, requires a better understanding of nitrogen (N) dynamics. Thus, for container E. globulus, considered one of the most important plantation species worldwide, we evaluated the effects of increasing N supply (50 to 600 mg N L−1) on seedling morphological traits, biomass production, root growth potential, and foliar N. After 15 weeks of N application, seedlings showed an increase in all growth variables evaluated up to 300 mg N L−1, whereas higher rates allowed luxury consumption. Modeling growth responses revealed, however, an apparent ammonium antagonism with other macronutrient cations suggests that high rates of this N-form may negate the potential benefits of N-loading, or that other modifications to the fertilizer solution may promote additional growth at higher N rates. Foliar N concentration, which was easily and accurately measured with a leaf chlorophyll meter, showed a gradient from basal to apical leaves suggesting a strong translocation of N in E. globulus seedlings. Our results suggest nursery management that supplies 300 mg L−1 of N through the entire growing season could improve seedling nutritional status, morphological attributes, and growth of new roots, attributes that may improve establishment on low fertility sites.

Root Penetration of Polyvinyl Chloride (PVC) Stormwater and Sewer Pipes

Two experiments investigated factors influencing root penetration of polyvinyl chloride (PVC) pipes. Eucalyptus leucoxylon, Allocasuarina littoralis, Lophostemon confertus, Callistemon salignus, Acer palmatum, and Pyrus calleryana seedlings were grown in containers containing 150-mm lengths of sealed 75-mm PVC stormwater pipe with cracks 0.04 mm, 0.66 mm, or 1.48 mm wide on their upper surface. The buried pipes contained water, water and stormwater, soil, or soil and stormwater. There were six replicates and 432 plants. There was no significant difference in the mass of roots entering the pipes for the two larger crack widths with 70% of pipes penetrated and strong growth inside the pipes. While the roots of all species penetrated cracks greater than 0.66 mm, only roots of C. salignus, E. leucoxylon, and L. confertus penetrated 0.04 mm cracks. Roots penetrated 50 to 60% of pipes containing soil, or soil and stormwater, and 40% of pipes containing water, or water and stormwater were penetrated. The plants with roots penetrating pipes containing water and stormwater grew tallest. No roots penetrated the welded caps of the stormwater pipes. A second experiment using E. leucoxylon, Melaleuca ericifolia, Ficus macrophylla, A. littoralis, and Salix fragilis investigated root penetration of different sized holes in polycarbonate plates. The plates, installed in containers with growing medium above and below, had either 2 × 4 mm holes, 8 × 2 mm holes, 127 × 0.5 mm holes, or a mixture of holes (1 × 4 mm, 2 × 2 mm and 32 × 0.5 mm holes), total pore area in all being 25.14 mm2. Below the plates, the growing medium was capillary irrigated with stormwater or water. All species grew through 0.5-mm holes and had strong root growth below the plates. When irrigated with stormwater, all species were taller and had greater biomass, and most species had a greater root mass below the plates. In general and regardless of hole size, the more holes in the plates, the more roots penetrated them. Properly installed PVC pipes are impenetrable, but the width and number of openings in a pipe influence the capacity for penetration and subsequent root growth so protocols minimizing damage to pipes should be enforced. Since species have different capacities for penetrating stormwater pipes, appropriate species selection for urban environments where damaged pipes may occur could reduce incidences of pipe damage.

The influence of inorganic salts, sucrose and CEPA on rooting of ujilloir cuttings

Hardwood or leafless softwood cuttings of <i>Salix viminalis gallica</i> (hort.) or <i>S. daphnoides acutifolia</i> Will, were soaked in water or separately in IAA, IBA, NaCl, KCl, NaNO<sub>3</sub>, KNO<sub>3</sub>, and sucrose solution or in sequence with one of the auxins, or CEPA. The inorganic salt treatments (KCl, KNO<sub>3</sub>, NaNO<sub>3</sub>) stimulated rooting of cuttings to the same degree as auxin, while sucrose and NaCl with IAA resulted in high stimulation of rooting, but with IBA no synergistic effect was observed in the other willow species. CEPA induced the largest increase of root number per cutting, stimulated significantly subsequent root growth and also reduced significantly the rooting period.

Improvement of the chilling tolerance of ‘Monika’ cucumber seedlings by short-term temperature and seed conditioning with plant growth regulators

ABSTRACT The aim of the present study was to improve the chilling tolerance of cucumber seedlings. The conditioned seeds in solutions of salicylic or jasmonic acid in concentrations of 10-2, 10-3 or 10-4 M or brassinolide in concentrations of 10-6, 10-8 or 10-10 M were subjected to temperature shock of 0, 2.5, 5, 35, 40 or 45°C for 1, 2 or 4 hours. Seedlings with 3 mm roots were chilled at 0°C for three days. The chilling susceptibility was evaluated by measurements of roots lengths, electrolyte leakage and total dehydrogenase activity. The obtained results indicated that cucumber tolerance to chilling conditions depended on the plant growth regulators used during seed imbibition and its concentration as well as the temperature initiating thermal shock and its duration. The highest tolerance to chilling, expressed by the subsequent growth of roots, was observed after seed conditioning in salicylic acid solutions in a concentration of 10-4 M for 16 h at 25°C, then exposed for 4 h to a shock temperature of 5°C. The length of seedling roots after such treatment was over 12 times longer than the control (imbibed in distilled water and not subjected to short-term temperature impact). An increased chilling tolerance of cucumber seedlings may result from enhanced membrane integrity and total dehydrogenase activity. Further research is needed to explain the mechanism of the positive effects of salicylic acid, jasmonic acid or brassinolide application to reduce the injurious effects of chilling on cucumber seedlings.

Auxins or Sugars: What Makes the Difference in the Adventitious Rooting of Stored Carnation Cuttings?

Cold storage of cuttings is frequently applied in the vegetative propagation of ornamental plants. Dianthus caryophyllus was used to study the limiting influences of auxin and sugars on adventitious root formation (ARF) in cuttings stored at 5°C. Carbohydrate levels during storage were modulated by exposing cuttings to low light or darkness. The resulting cuttings were treated (or not) with auxin and planted, and then ARF was evaluated. Carbohydrate levels in the cuttings were monitored and the influence of light treatment on indole-3-acetic acid (IAA) and zeatin (Z) in the basal stem was investigated. Dark storage for up to 4 weeks increased the percentage of early rooted cuttings and the final number and length of adventitious roots, despite decreased sugar levels in the stem base. Light during cold storage greatly enhanced sugar levels, particularly in the stem base where the Z/IAA ratio was higher and ARF was lower than observed in the corresponding dark-stored cuttings. Sugar levels in nonstored and dark-stored cuttings increased during the rooting period, and auxin application enhanced the accumulation of sugars in the stem base of nonstored cuttings. Auxin stimulated ARF most strongly in nonstored, less so in light-stored, and only marginally in dark-stored cuttings. A model of auxin-sugar interactions in ARF in carnation is proposed: cold storage brings forward root induction and sink establishment, both of which are promoted by the accumulation of auxin but not of sugars, whereas high levels of sugars and probably also of cytokinins act as inhibitors. Subsequent root differentiation and growth depend on current photosynthesis.

Transcriptome responses to aluminum stress in roots of aspen (Populus tremula)

BackgroundIonic aluminum (mainly Al3+) is rhizotoxic and can be present in acid soils at concentrations high enough to inhibit root growth. Many forest tree species grow naturally in acid soils and often tolerate high concentrations of Al. Previously, we have shown that aspen (Populus tremula) releases citrate and oxalate from roots in response to Al exposure. To obtain further insights into the root responses of aspen to Al, we investigated root gene expression at Al conditions that inhibit root growth.ResultsTreatment of the aspen roots with 500 μM Al induced a strong inhibition of root growth within 6 h of exposure time. The root growth subsequently recovered, reaching growth rates comparable to that of control plants. Changes in gene expression were determined after 6 h, 2 d, and 10 d of Al exposure. Replicated transcriptome analyses using the Affymetrix poplar genome array revealed a total of 175 significantly up-regulated and 69 down-regulated genes, of which 70% could be annotated based on Arabidopsis genome resources. Between 6 h and 2 d, the number of responsive genes strongly decreased from 202 to 26, and then the number of changes remained low. The responses after 6 h were characterized by genes involved in cell wall modification, ion transport, and oxidative stress. Two genes with prolonged induction were closely related to the Arabidopsis Al tolerance genes ALS3 (for Al sensitive 3) and MATE (for multidrug and toxin efflux protein, mediating citrate efflux). Patterns of expression in different plant organs and in response to Al indicated that the two aspen genes are homologs of the Arabidopsis ALS3 and MATE.ConclusionExposure of aspen roots to Al results in a rapid inhibition of root growth and a large change in root gene expression. The subsequent root growth recovery and the concomitant reduction in the number of responsive genes presumably reflect the success of the roots in activating Al tolerance mechanisms. The aspen genes ALS3 and MATE may be important components of these mechanisms.

Involvement of root ethylene and oxidative stress-related activities in pre-conditioning of tomato transplants by increased salinity

SummaryIn order to understand the mechanism(s) of pre-conditioning (PC) by increased levels of salinity that enhance transplant establishment in the field, we studied the possible roles of ethylene and oxidative stress in transplants of tomato (Solanum lycopersicum L.) during PC by saline treatments. Prior to transplanting, transplants were fed hydroponically for 6 d with a normal nutrient solution, or solutions containing various concentrations of NaCl, ethephon, or silver thiosulphate (STS), or various combinations of these chemicals. The transplants were then transferred to normal nutrient solution for 10 d to recover. Addition of up to 75 mM NaCl to the nutrient solution decreased root elongation slightly and significantly increased lateral root (LR) formation during PC, but enhanced the subsequent recovery of roots and shoots. All the salinity treatments inhibited root ethylene production. However, the application of 20 mg l–1 ethephon to the roots during PC increased ethylene concentrations, inhibited root elongation, increased LR formation, and stimulated subsequent root growth.An enhancement of root growth during recovery was also observed when 75 mM NaCl plus 20 mg l–1 ethephon was applied to the root system during PC. The ethylene blocker, STS, enhanced root elongation during PC, and acted synergistically with NaCl in enhancing subsequent root growth. As PC treatments, NaCl or ethephon both increased root malonyldialdehyde (MDA) and H2O2 concentrations, and increased root peroxidase and superoxide dismutase (SOD) activities; but, if NaCl or ethephon were combined with STS, the extent of these increases was diminished. Application of ≤ 1 mM H2O2 at PC increased subsequent root and shoot growth in seedlings, but higher H2O2 concentrations were inhibitory. These results suggest that subsequent root growth upon recovery from transplantation benefits from PC applications of NaCl or ethephon and this might be due the enhancement of LR initiation during PC by these treatments, possibly through the signalling role of H2O2. STS alleviates oxidative stress in the root by inhibiting the action of ethylene and decreasing ethylene sensitivity, thereby promoting root recovery. These morphological and biochemical changes may explain the beneficial effect of PC on transplant development under field conditions.

Tissue culture of Sinningia speciosa and analysis of the in vitro-generated tricussate whorled phyllotaxis (twp) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l−1 6-benzylaminopurine (BA) and 0.2 mg l−1 α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l−1 NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.

Involvement of nitric oxide-induced NADPH oxidase in adventitious root growth and antioxidant defense in Panax ginseng

Nitric oxide (NO) affects the growth and development of plants and also affects plant responses to various stresses. Because NO induces root differentiation, we examined whether or not it is involved in increased ROS generation. Treatments with sodium nitroprusside (SNP), an NO donor, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a specific NO scavenger, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an NO synthase (NOS) inhibitor, revealed that NO is involved in the adventitious root growth of mountain ginseng. Supply of an NO donor, SNP, activates NADPH oxidase activity, resulting in increased generation of O2·−, which subsequently induces growth of adventitious roots. Moreover, treatment with diphenyliodonium chloride (DPI), an NADPH oxidase inhibitor, individually or with SNP, inhibited root growth, NADPH oxidase activity, and O2·− anion generation. Supply of the NO donor, SNP, did not induce any notable isoforms of enzymes; it did, however, increase the activity of pre-existing bands of NADPH oxidase, superoxide dismutase, catalase, peroxidase, ascorbate peroxidase, and glutathione reductase. Enhanced activity of antioxidant enzymes induced by SNP supply seems to be responsible for a low level of H2O2 in the adventitious roots of mountain ginseng. It was therefore concluded that NO-induced generation of O2·− by NADPH oxidase seems to have a role in adventitious root growth of mountain ginseng. The possible mechanism of NO involvement in O2·− generation through NADPH oxidase and subsequent root growth is discussed.

Bio-stimulatory properties in seeds of plants from the families Caryophyllaceae and Fabaceae with application potential in agriculture

A comparative investigation into the bio-stimulatory activity of seed suspensions from two South African plant species belonging to each of the families Caryophyllaceae and Fabaceae were conducted using ComCat®, a commercially available bio-stimulant, as a positive control. These included Acacia erioloba and Acacia karroo from the family Fabaceae and Dianthus basuticus and Pollichia campestris from the family Caryophyllaceae. Three different bio-assays were applied to screen for possible bio-stimulatory activity in the seed suspensions. These included the effect on the respiration rate of monoculture yeast cells, germination of seeds from five vegetable crops and subsequent root growth in seedlings. The study confirmed that seed suspensions of A. eri-oloba and P . c a m p e s tr is were consistently most active in terms of all three bio-assays used to test for bio-stimulatory activity. Following liquid-solid extraction of crude extracts from these two species by using organic solvents with increasing polarity, a very small amount of active extract was recovered from P. campestris seeds. Subsequently, only the semi-purified and active ethyl acetate fraction of an A. erioloba crude extract was purified further. By means of column- and preparative thin layer chromatography the active compound was isolated, purified and identified as 2-ethyl-hexyl phthalate by means of nuclear magnetic resonance (NMR) spectroscopy. The latter was proven not to be an artefact of the extraction process. It is concluded that sufficient evidence in terms of the application potential of an A. erioloba extract in agriculture has been produced to warrant further investigation under field conditions.

Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology

We report the production of tetraploid plants of Platanus acerifolia, with the ultimate aim of improving the ornamental qualities of this important urban landscaping tree. Chromosome doubling was achieved by the application of colchicine to either pre-soaked seed or to the apical meristems of young seedlings. Treatment of the ungerminated seed was the more efficient method in terms of numbers of tetraploid seedlings (up to 40%, as determined by chromosome counting of the root-tip nuclei) but this method produced no mature tetraploid plants due to the deleterious effect of colchicine on subsequent root growth. When colchicine was applied directly to the apical growing tip of cotyledon-stage seedlings, leaf and stem growth was temporarily affected but the plants eventually recovered. We conducted a preliminary screen for putative tetraploids based on the observation in other plant species of a correlation of stomatal size and distribution with ploidy. Plants containing significantly larger stomata and at a lower density across the lower leaf epidermis, were selected for further analysis by flow cytometry and chromosome counting. These techniques confirmed that, of the 12 putative polyploids, four were tetraploid, five were mixoploid and three were, in fact, diploid. Morphological differences of the tetraploids included a more compact growth habit and broader, thicker leaves. These plants are being grown to full maturity in order to test their potential for use in a breeding programme aimed at producing sterile triploid lines.

Lignosulfonate promotes the interaction between Scots pine and an ectomycorrhizal fungus Pisolithus tinctorius in vitro

Lignosulfonate (LS) is a lignin-based polymer obtained as a by-product from paper industry, which may have potential as an amendment with macronutrients. We studied effects of LS on the interaction between Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings and the ectomycorrhizal (ECM) fungusPisolithus tinctorius (Pers.) Coker and Couch. The experiments were performed in vitroon the MMN agar medium containing Fe–LS chelate at the concentrations of 0, 5, 10 and 25 mg/L. Inoculation with P. tinctoriusincreased root growth of the seedlings. Fe–LS enhanced P. tinctorius induced formation of lateral roots and had a dose-dependent positive effect on the establishment of mycorrhizas on the seedlings. The growth of the fungal mycelium was improved by Fe–LS, which might cause faster and more intensive contact with the roots and, thus, better root growth and mycorrhiza formation. P.␣tinctorius enhanced also adventitious root formation and subsequent root growth of the hypocotyl cuttings but without any synergistic effect with Fe–LS. Our study with P. tinctorius and Scots pine in vitro indicates that a low-cost by-product Fe–LS, obtained from paper industry, may be a potential tool to improve the efficiency of fungal inoculations, thus, facilitating the early interaction between an ECM fungus and host seedling.

Light sources with different spectra affect root and mycorrhiza formation in Scots pine in vitro

We studied the effects of broad-spectrum light quality on the interaction between the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch and Scots pine (Pinus sylvestris L.) seedlings and hypocotyl cuttings cultured in vitro. The light sources were cool white (CW), warm white (WW) and red-rich daylight (RD) fluorescent lamps. Inoculation with P. tinctorius enhanced adventitious root formation of the cuttings in all light treatments. Rooting of the inoculated cuttings was highest in WW light (89%), followed by CW (73%) and RD light (66%). During 6 weeks of in vitro culture, rooted cuttings formed only a few lateral roots. The fungus grew over lateral roots, but the Hartig net was absent in all light treatments. In non-inoculated cuttings, neither root formation nor subsequent root growth was affected by light quality. In the seedling experiment, inoculation in the WW treatment resulted in a significantly (P < 0.05) greater number of lateral roots than inoculation in the RD treatment. The percentage of lateral roots covered with fungal hyphae was also highest in WW light (62%), followed by CW (50%) and RD (27%) light. A similar pattern was observed in the intensity of Hartig net formation. We conclude that effects of broad-spectrum light quality on the ectomycorrhizal fungus-root interaction are dependent on the developmental stage of the root.

Application of Ectomycorrhizal Fungi in Vegetative Propagation of Conifers

In forestry, vegetative propagation is important for the production of selected genotypes and shortening the selection cycles in genetic improvement programs. In vivo cutting production, in vitro organogenesis and somatic embryogenesis are applicable with conifers. However, with most coniferous species these methods are not yet suitable for commercial application. Large-scale production of clonal material using cuttings or organogenesis is hindered by rooting problems and difficulties in the maturation and conversion limit the use of somatic embryogenesis. Economically important conifers form symbiotic relationship mostly with ectomycorrhizal (ECM) fungi, which increase the fitness of the host tree. Several studies have shown the potential of using ECM fungi in conifer vegetative propagation. Inoculation with specific fungi can enhance root formation and/or subsequent root branching of in vivo cuttings and in vitro adventitious shoots. Germination of somatic embryos and subsequent root growth can also be improved by the use of ECM fungi. In addition, inoculation can increase the tree's ability to overcome the stress related to ex vitro transfer. A specific interaction between a fungal strain and tree clone occurs during root induction and germination of somatic embryos. Multiple rooting factors exist in this interaction that complicate the predictability of the response to inoculation. Fungal-specific factors that influence rooting responses to inoculation may include plant growth regulator production, modification of the rooting environment, and interactions with beneficial microbes. A combination of these factors may act synergistically to result in positive responses in tree genotypes that are compatible with the fungus.

Effects of exogenous diamines on the interaction between ectomycorrhizal fungi and adventitious root formation in Scots pine in vitro.

Production of free and conjugated polyamines by two ectomycorrhizal fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Paxillus involutus (Batsch) Fr., was studied in vitro. Spermidine was the main polyamine in the mycelium of both fungi. Paxillus involutus also produced large amounts of the diamine putrescine, whereas Pisolithus tinctorius contained traces of the diamine cadaverine and released into the culture medium an unknown compound probably related to cadaverine or N-methylputrescine. Both fungi accelerated adventitious root formation and increased subsequent root growth of Scots pine (Pinus sylvestris L.) hypocotyl cuttings in vitro. Exogenous cadaverine enhanced rooting caused by Pisolithus tinctorius and also promoted mycorrhiza formation by the fungus. Putrescine and Paxillus involutus had a synergistic effect on root initiation, but not on subsequent root growth. We conclude that specific diamines may be involved in the interaction between ectomycorrhizal fungi and adventitious root formation in Scots pine, and that the effects of specific exogenous polyamines are dependent on the fungal strain and its ability to produce these compounds. The finding that Paxillus involutus enhanced rooting and root growth without mycorrhiza formation indicates that fungal-induced rooting is not necessarily related to visible mycorrhiza formation.