The ectomycorrhizal basidiomycete Tricholoma matsutake associates with members of the Pinaceae such as Pinus densiflora (red pine), forming a rhizospheric colony or "shiro," which produces the prized "matsutake" mushroom. We investigated whether the host specificity of T. matsutake to conifers is innately determined using somatic plants of Cedrela odorata, a tropical broad-leaved tree (Meliaceae) that naturally harbors arbuscular mycorrhizal fungi. We found that T. matsutake could form in vitro shiro with C. odorata 140 days after inoculation, as with P. densiflora. The shiro was typically aromatic like that of P. densiflora. However, this was a root endophytic interaction unlike the mycorrhizal association with P. densiflora. Infected plants had epidermal tissues and thick exodermal tissues outside the inner cortex. The mycelial sheath surrounded the outside of the epidermis, and the hyphae penetrated into intra- and intercellular spaces, often forming hyphal bundles or a pseudoparenchymatous organization. However, the hyphae grew only in the direction of vascular bundles and did not form Hartig nets. Tricholoma fulvocastaneum or "false matsutake" naturally associates with Fagaceae and was also able to associate with C. odorata as a root endophyte. With T. matsutake, C. odorata generated a number of roots and showed greatly enhanced vigor, while with T. fulvocastaneum, it generated a smaller number of roots and showed somewhat lesser vigor. We argue that the host-plant specificity of ectomycorrhizal matsutake is not innately determined, and that somatic arbuscular mycorrhizal plants have a great potential to form mutualistic relationships with ectomycorrhizal fungi.
Somatic embryogenesis (SE) in not only one of the most promising techniques for mass propagation of selected trees, but also is a valuable tool for basic research studies in cell biology and genetic engineering, and it allows the long-term ex situ conservation of genetic resources by cryopreservation techniques. This review reports the most recent progress in SE, protoplast culture, and cryopreservation of four important Japanese pines (Pinus thunbergii, Pinus densiflora, Pinus armandii var. amamiana, and Pinus luchuensis). Induction of embryogenic tissues (ET), embryogenic culture maintenance/proliferation, production of somatic embryos, germination, and conversion to plants are described focusing on the protocols most commonly reported for plant production in Pinus species through to SE.
We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or "shiro". We questioned whether this phenomenon could have occurred because of plant-microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant-fungal symbiosis.
One of the possible countermeasures for pollinosis caused by sugi (Cryptomeria japonica), a serious public health problem in Japan, is the use of male sterile plants (MSPs; pollen-free plants). However, the production efficiencies of MSPs raised by conventional methods are extremely poor, time consuming, and resulting in a high seedling cost. Here, we report the development of a novel technique for efficient production of MSPs, which combines marker-assisted selection (MAS) and somatic embryogenesis (SE). SE from four full sib seed families of sugi, carrying the male sterility gene MS1, was initiated using megagametophyte explants that originated from four seed collections taken at one-week intervals during the month of July 2017. Embryogenic cell lines (ECLs) were achieved in all families, with initiation rates varying from 0.6% to 59%. Somatic embryos were produced from genetic marker-selected male sterile ECLs on medium containing maltose, abscisic acid (ABA), polyethylene glycol (PEG), and activated charcoal (AC). Subsequently, high frequencies of germination and plant conversion (≥76%) were obtained on plant growth regulator-free medium. Regenerated plantlets were acclimatized successfully, and the initial growth of male sterile somatic plants was monitored in the field.
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