Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild-type and indoleacetic acid (IAA)-overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA-overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster . No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.     

Gibberellin A3 stimulates adventitious rooting of cuttings from cherry (Prunus avium)

We have examined the effect of exogenous gibberellin A₃(GA₃) on adventitious rooting of Prunus avium(cherry) cultivars Stella, F12/1 and Charger. We show that GA₃pre-treatment of P. avium stock plants causes an increasein shoot growth rate and also improves the rooting of cuttings subsequentlytaken from the treated plants. Approximately 37% of cuttings from controlshootsrooted, whereas the percentage rooting could be increased to 80% or more withGA₃ pre-treatment. The number of roots per rooted cutting was alsoincreased by GA₃ pre-treatment. The stimulation of adventitiousrooting could be partially explained by the increase in shoot growth rate.Cultivar Charger responded better than the other cultivars to the lowest levelof GA₃ treatment. In vitro cultures of cultivarCharger were also treated with GA₃. However, the stimulation ofadventitious rooting was less marked than in the GA₃-treated stockplants: percentage rooting increased from 70% to 85%. The results are discussedin the context of 'rejuvenation' effects of GA₃.     

Influence of Sugars on in vitro Rooting and Acclimatization of Carob Tree

Carob tree (Cerafoma siliqua L.) micropropagated shoots were rooted on half-strength Murashige and Skoog medium, supplemented with different types and concentrations of sugars, in order to determine the effects of sugar composition and concentration on in vitro rooting and in vivo establishment of the plantlets. Among the various sugars tested, the best rooting response was obtained with 145 mM sucrose, both in terms of rooting frequency and index of rooting. The use of filter-sterilized rather that autoclaved fructose increased root number and root length. Sugar treatment during rooting slightly influenced plantlet survival and growth during acclimatization. A reduction in the glucose concentration during rooting was beneficial for plantlet acclimatization.     

Root production and plantlet development in tissue-cultured conifers

Rooting and acclimatization procedures for micropropagated conifers are reviewed, with emphasis on their effects on root quality and plantlet performance in the nursery and field. Major influences on root production include auxin concentration and mode of application, shoot quality, donor age, clone and temperature. The development of a fibrous, well-branched root system has been a problem that may be solved by using rooting substrates that are better-aerated than agar. Further development of the root system may be enhanced by early air-pruning and ectomycorrhizal associations. During acclimatization, high humidity is required for conifers. However, conifers have an advantage over non-coniferous plantlets with respect to water loss because of a better development of the needle cuticles prior to transfer to in vivo conditions. In greenhouse and field comparisons with seedlings, plantlets were similar in survival and growth rate, but root systems were less fibrous. Also, features of early maturation have been observed for plantlets, the cause of which is uncertain. Pertinent research with rooted cuttings and seedlings of conifers has been cited to gain a better understanding of the factors involved in root production and development.     

Vegetation influence on ectomycorrhizal inoculum Available to sub-arctic willow (Salix lapponum L.) planted in an upland Site

Summary

Restoration of scrub and woodland in deforested upland sites is an important conservation activity. However, little is known about the mycorrhizal colonisation potential of upland soils or the factors that influence the distribution of mycorrhizal inoculum. We investigated the effect of existing vegetation on mycorrhizal colonisation potential for a sub-arctic willow (Salix lapponum) by planting uninoculated cuttings into plotsrepresenting two upland habitats with either grassand herbs (‘grass’) or Vaccinium myrtillus (‘vaccinium’) and assessing mycorrhizal colonisation after 14 months using morphological and molecular techniques. From 40 willow cuttings (20 in each habitat), DNA sequences of rive ectomycorrhizal (EcM) fungal taxa were recovered: Laccaria proxima, Thelephora terrestris, Hebeloma sp., ‘Thelephoraceae sp.’ and ‘Pezizales sp.’. Cuttings in the ‘grass’ habitat were dominated by Laccaria proxima and ‘Pezizales sp.’ and in the ‘vaccinium’ habitat by Thelephora terrestris which was absent from the ‘grass’ habitat. There were no significant differences between habitats in frequency of EcM inoculum (overall percentage of cuttings colonised = 70%) or colonisation potential (overall mean percentage of root tips colonised per cutting = 20 %). These data suggest that the mycorrhizal colonisation potential and diversity of fungi available to willow in these upland soils are low and planted willow may benefit from inoculum enhancement.     

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

In vitro responses and acclimatization of Prunus serotina with paclobutrazol

Paclobutrazol (PBZ), a triazole growth retardant known to improve tolerance of various species to stress, was incorporated into the in vitro rooting medium of Prunus serotina var. virens at rates of 0.00, 0.15, 0.30, and 0.60 mg/L with and without 1.0 mg/L indolebutyric acid (IBA). PBZ significantly reduced shoot growth in vitro but increased/improved the quality and coloration. The percentage of water loss from detached leaves of in vitro plantlets was significantly reduced by PBZ and IBA. At 4 weeks after transfer to the greenhouse, survival was significantly improved by PBZ, IBA, and the combination. Incorporation of PBZ in vitro better enables Prunus serotina plantlets to withstand the stresses associated with acclimatization.     

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.     

Development of mycorrhized vitroplants of Jatropha curcas L. at different rooting stages

In this work, we attempted to assess the effects of inoculation of arbuscular mycorrhizal fungus (AMF), Glomus clarum, on the survival and development of micropropagated Jatropha curcas plantlets at different rooting stages. Elongated shoots (average 3?cm) of J. curcas, maintained for 0, 14, or 21?days on rooting medium in the presence or absence of 1?mg?L^?1 indole-3-butyric acid (IBA), were transferred to a sand:soil:vermiculite (1:1/2:1) (v:v:v) substrate. At the time of transplantation, the plantlets were either inoculated or not inoculated with G. clarum that had been monoaxenically produced in Ri-transformed carrot roots. After a 2-week acclimatization period, 100?% of the plants kept for 0 or 14?days in rooting medium survived. However, those that remained for 21?days in rooting medium displayed post-acclimatization survival rates of 93 and 89?% for plants inoculated and non-inoculated with G. clarum, respectively. Colonization rates ranged from 70 to 93?%, and the stimulatory effects of AMF were evidenced by increased phosphorus uptake by plants and increases in all evaluated growth parameters, except plant height. Plants that were not subjected to the rooting stage showed growth similar to or higher than those subjected to the rooting stage, regardless of the addition of IBA. It can be concluded that stimulatory effects of mycorrhizal fungi were observed, and that the inoculation of J. curcas proved effective during the initial period of the acclimatization phase.     

In vitro propagation of a semi-dwarfing cherry rootstock

A successful in vitro propagation system for the semi-dwarfing cherry rootstock Maxma-14 (Prunus avium L.) has been developed. Shoot tips and axillary buds were successfully established in vitro. Multiplication rate of about 6 was achieved over a 4-week period using Murashige and Skoog medium with 4.44 μM benzyladenine and 0.49 μM indole-3-butyric acid (IBA). Rooting occurred within 4 weeks on liquid and agar-gelled media containing 0.49 μM NAA or 0.49, 2.45 μM IBA. On liquid media, a maximum rooting efficiency of up to 100% was obtained. However, high concentrations of auxins delayed the time of root initiation for 3–5 days. Acclimatization was affected directly by rooting conditions. Survival was best when plantlets were transferred to pots after a short period of root emergence on rooting media. Multiplication medium was also important for successful acclimatization. Shoots transferred to rooting media from that with kinetin resulted in better acclimatization and survival than that derived from media with benzyladenine. Further, plantlets rooted on liquid media had better survival than that rooted on agar-gelled media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of isolates of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens that were dominant in soil from disturbed sites on growth of Betula platyphylla var. japonica seedlings

Effects of ectomycorrhizal fungi and endophytic Mycelium radicis atrovirens Melin (MRA) on growth of Betula platyphylla var. japonica seedlings were investigated under aseptic culture conditions. Three isolates of ectomycorrhizal fungi and two isolates of MRA were used. One MRA isolate was Phialocephala fortinii. Previous field work revealed that these isolates were dominant on the roots of B. platyphylla var. japonica seedlings grown in a mineral subsoil that had been exposed by the removal of surface soil. After a 100-day incubation, the growth of the seedlings was significantly enhanced by the colonization of these ectomycorrhizal fungal isolates as compared with uninoculated seedlings. In contrast, the growth of seedlings was retarded by the colonization of the MRA isolates. The growth of seedlings that were co-inoculated with ectomycorrhizal fungi and MRA was similar to that of uninoculated seedlings in most cases. These results suggest that ectomycorrhizal fungi have a beneficial effect on the growth of B. platyphylla var. japonica seedlings and that they suppress the deleterious effect of MRA. Thus, these ectomycorrhizal fungi probably have an important role in establishing B. platyphylla var. japonica seedlings during the initial stage of re-vegetation following site disturbance by the removal of surface soil.     

Mycorrhizal effects on the acclimatization, survival, growth and chlorophyll of micropropagated Syngonium and Draceana inoculated at weaning and hardening stages

 Micropropagated plantlets of Syngonium podophyllum and Draceana sp. were inoculated during an early weaning stage of acclimatization with a mixed indigenous consortium of arbuscular mycorrhizal (AM) fungi. Both species were colonized but a significantly higher colonization was observed (38%) in Draceana than S. podophyllum when it was harvested after 20 weeks. Draceana plants showed little difference in the extent to which they were colonized, when examined either at the weaning stage or hardening stage; however, S. podophyllum plants at the weaning stage were better colonized than at 20 weeks. Survival was high in inoculated plants at lower fertility in both hosts. Moreover, S. podophyllum showed better stolon production than uninoculated controls at both fertility levels, though the increase was higher at lower fertility. Draceana showed no response in shoot height to any treatment. Chlorophyll accumulation in both hosts was significantly influenced by inoculation, fertility and stage (weaning and hardening). A significant increment in shoot P uptake was also observed in both hosts related to inoculation, stage and fertility. Inoculation with the AM consortium had a significant and favourable effect on acclimatization of micropropagated S. podophyllum and Draceana at the weaning stage, saving almost 15 days in the total hardening process. Received: 13 August 1998 / Accepted: 25 August 1999     

Delayed flowering and reduced branching in micropropagated mature wild cherry (Prunus avium L.) compared with rooted cuttings and seedlings

The development of micropropagated wild cherry (Prunus avium L.) was compared in the nursery and the field with cuttings and seedlings, over a total period of 6 years. Summer semi-hardwood cuttings tended to produce moderate numbers of branches in the season following propagation, whereas micropropagules and seedlings produced significantly fewer or none at all. Removal of branches from cuttings resulted in taller trees. A greater proportion of cuttings than micropropagules flowered in the first year in which trees produced flowers. In the second flowering year, there were no differences in flowering habit between cuttings and micropropagules. Propagation by cuttings or micropropagation did not consistently affect increments in stem diameters or heights. These results are discussed in terms of the suitability of micropropagation and cuttings to produce clonal wild cherry. Received: 23 February 1997 / Revision received: 3 January 1998 / Accepted: 5 February 1998     

Micropropagation of Naregamia alata W & A-An Important Medicinal Plant

In vitro clonal multiplication of a valuable medicinal species, Naregamia alata is reported. In shoot tip cultures, maximum multiplication (9 fold/28 days) occurred on MS medium containing 3.1 μM 6-benzylaminopurlne and 8.6 μM gibberellic acid. For the rooting of the shoots, 9.8 μM indole 3-butyric acid was the best. After acclimatization, 90% of the micropropagated plantlets were successfully established in the field.     

Abscisic Acid in Prunus Trees: Isolation and the Effect on Growth of Excised Shoot Tissue

The abscisic acid content of the mature shoot tissue of vigorous Prunus avium, medium sized Prunus cerasus and dwarfing Prunus cerasus self (S) was estimated by spectropolarimetry and gas chromatography. There is no correlation between the levels of ABA found in the tissue and the dwarfing potential of the trees investigated. However, Prunus arium stem tissue shows the lowest sensitivity to ABA, whereas the explants of normal and dwarfing Prunus cerasus exhibit a stronger inhibition.     

Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings

Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.Abbreviations BA Benzyl-Aminopurine - NAA Naphthalene Acetic Acid - IBA Indole-3-Butyric Acid - MS medium Murashige and Skoog's medium - DBH Diameter at Breast Height     

Response of ectomycorrhizal fungi to benomyl and nocodazole: growth inhibition and microtubule depolymerization

The growth of seven ectomycorrhizal fungi was tested in the presence of the antimicrotubule drugs benomyl and nocodazole. The polymerization stage of the cytoplasmic microtubules in the hyphal cells was visualized by indirect immunofluorescence microscopy after a 3-h drug treatment. Nocodazole reduced the growth of all the fungi tested at concentrations of 2 and 4 g ml^-1 and caused strong depolymerization of microtubules in all other species except Hebeloma cylindrosporum. Benomyl inhibited the growth and depolymerized the microtubules in the ascomycete Cenococcum geophilum, while in the basidiomycetes it reduced the growth and depolymerized the microtubules only in H. cylindrosporum. The role of the microtubule cytoskeleton and the target of the benzimidazole-derived drugs in fungal cells are discussed.     

Assessing past agrobiodiversity of Prunus avium L. (Rosaceae): a morphometric approach focussed on the stones from the archaeological site H?tel-Dieu (16th century, Tours, France)

Abundant and diverse Prunus fruitstone remains from cherries, plums, sloes, peaches, etc. are frequently recovered from archaeological waterlogged contexts such as wells, latrines, lake dwellings etc. in Europe. The distinction between most of the Prunus species, based on traditional morphological characters of the fruit stones, is usually not problematic. However the discrimination between P. avium L., P. cerasus L. and related cherry species, based on classical criteria alone, often turns out to be ambiguous because of the increasing number of varieties which have been bred since Roman times. By combining geometric and traditional morphometrical approaches, the overall variation in shape and size of stones from French and Swiss excavations dating from the 1st century to the 16th century a.d. were assessed. Among these important archaeobotanical data, the detailed examination of 100 waterlogged stones from the 16th century H?tel-Dieu cesspit at Tours, France, revealed that the morphological diversity is structured into two distinct morphotypes which diverge mainly according to geometrical features. Finally, the comparison between morphological features of these well-preserved archaeological stones and modern reference material including P. avium, P. cerasus and P. × gondouinii, suggests that these two morphotypes, which have been initially attributed to P. avium (long stones) and P. avium/cerasus (rounded stones) according to traditional morphological parameters, would correspond to two different cultivated varieties, both belonging to Prunus avium. Results presented in this work constitute new and preliminary data obtained during the development of this project that throw light on morphological variability and biosystematic aspects.     

Arbuscular mycorrhizal (AM) technology for the conservation of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn.: an endangered medicinal herb

Curculigo orchioides Gaertn. (family Hypoxidaceae) is an endangered anticarcinogenic and aphrodisiac herb, native of India. This study reports the effect of three arbuscular mycorrhizal (AM) fungal inocula on post-transplanting performance of ‘in vitro’ raised C. orchioides plantlets. The three AM fungal inocula consisted of two monospecific cultures of Glomus geosporum and G. microcarpum and one crude consortium of AM fungal spores isolated from rhizosphere soil of C. orchioides growing in natural habitat. Complete plantlets of C. orchioides were raised by direct organogenesis of leaf explants on half strength Murashige and Skoog’s medium devoid of any growth hormone. C. orchioides plantlets responded significantly different to all three mycorrhizal treatments. Mycorrhization enhanced the survival rate of C. orchioides plantlets to 100%. The inoculated plantlets fared significantly better than the uninoculated ones in terms of biomass production and number of leaves and roots per plant. Mycorrhizal plantlets exhibited higher concentrations of photosynthetic pigments as well as minerals P, Mg, Cu, Zn, Mn and Fe in both shoots and roots. Among the three inocula tested, plantlets inoculated with the mixed consortium of AM fungi consistently performed better in terms of the parameters evaluated. The study suggests use of mixed consortium of AM fungi over monospecific cultures for the sustainable cultivation and conservation of endangered medicinal plant: Curculigo orchioides.     

Microsatellite markers for Hebeloma species developed from expressed sequence tags in the ectomycorrhizal fungus Hebeloma cylindrosporum

The increasing number of expressed sequence tag (EST) projects dedicated to ectomycorrhizal fungi is translating into the release of large sets of ESTs. The aim of this study was to develop and test simple sequence repeat (SSR) markers from EST databases of the model ectomycorrhizal fungus Hebeloma cylindrosporum. Six SSR markers were found to be both unambiguously scorable and polymorphic among 12 H. cylindrosporum isolates. Two SSR markers were transferable to other Hebeloma species and one marker was interestingly found to be polymorphic among seven H. crustuliniforme isolates.