Ectomycorrhiza formation ofTricholoma matsutake onPinus densiflora

Mycorrhizal association ofTricholoma matsutake withPinus densiflora was studied. A naturally establishedP. densiflora stand (age: ca. 45 yr) where occurrences ofT. matsutake sporocarps had been confirmed was studied in lbaraki Prefecture, Japan. Pine root systems connected withT. matsutake sporocarps via the fungal white mycelia were sampled in October 1997. The sampled pine roots were covered overall with mycelia. Under a dissecting microscope, the mycelia were confirmed to form fungal sheaths on the lateral roots. Under a light microscope, transverse and longitudinal sections of these roots showed the presence of both fungal sheaths and Hartig nets, which are typical of ectomycorrhizas. The fungal sheath was ca. 1.5–20 μm. in thickness, and felt prosenchymatous in texture. Hartig nets developed continuously at the cortex and extended to the boundary between cortical cells and endodermal cells. The same ectomycorrhizal morphotype on the pine was also recovered from inside the same mycelial colony (i.e., “shiro”) ofT. matsutake from winter to summer. These results suggest thatT. matsutake has a perennial ectomycorrhizal association withP. densiflora.     

Mycorrhizal association of isolates from sporocarps and ectomycorrhizas withPinus densiflora seedlings

Thirty-two isolates from sporocarps of 27 species of macromycetes, 43 isolates from ectomycorrhizas ofPinus densiflora (Japanese red pine) and 1 isolate from an ectomycorrhiza ofQuercus myrsinaefolia were tested for the ability to form mycorrhizas withP. densiflora seedlings in glass tubes. Ten isolates from sporocarps ofHebeloma sp.,Laccaria bicolor, Lactarius chrysorrheus, Suillus granulatus, Scleroderma areolatum, Russula mariae andR. nigricans had formed ectomycorrhizas by 8 months after transplantation. Twenty isolates taken from mycorrhizas including ofCenococcum geophilum, R. mariae andR. nigricans formed ectomycorrhizas. The synthesized mycorrhizas were classified based on morphological characteristics such as hyphal arrangement of their fungal sheath, and appearance of cystidia and emanating hyphae. Twenty-one mycorrhizal types were recognized.Contribution No. 122, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Formation of Mycorrhiza-like Structures in Cultured Root/Callus of Cathaya argyrophylla Chun et Kuang Infected with the Ectomycorrhizal Fungus Cenococcum geophilum Fr.

An in vitro system was used for ectomycorrhizal synthesis of Cenococcum geophilum Fr. with Cathaya argyrophylla Chun et Kuang, an endangered species. Calli initiated from stem segments and adventitious roots differentiated from young seedlings were removed and cocultured with Cenococcum geophllum on a modified Murashlge-Skoog medium. Fungal hyphae were visible within intercellular spaces of the callus 4 weeks after inoculation, but definite and well-developed Hartig net structures did not form in the calU 8 weeks after inoculation. The typical ectomycorrhizal structures （i.e. hyphal mantle and Intracortical Hartig net） were observed in root segments 8 weeks after inoculation. This is the first report of aseptic ectomycorrhlzal-like formation/infection between root organ/callus of Cathaya argyrophylla and the ectomycorrhizal fungus Cenococcum geophflum. This culture system is useful for further investigation of mycorrhizal synthesis in Cathaya trees.     

Rapid in vitro ectomycorrhizal infection onPinus densiflora roots byTricholoma matsutake

The root systems of 11-wk-oldPinus densiflora seedlings were inoculated with a hyphal suspension ofTricholoma matsutake and aseptically incubated for 4 wk in a forest soil without supplying exogenous carbohydrates. One week following inoculation, fungal hyphae had colonized the root surface and bound soil particles together establishing a root-substrate continuum. Fungal hyphae were visible within the main root cortex following clearing bleaching and staining. In the ensuing days, fungal colonization was observed within elongating lateral roots in which Hartig net formation was confirmed 4 wk after inoculation. This is the first report of rapid ectomycorrhizal infection ofP. densiflora seedings byT. matsutake.     

Formation of Mycorrhiza-like Structures in Cultured Root/Callus of Cathaya argyrophylla Chun et Kuang Infected with the Ectomycorrhizal Fungus

An in vitro system was used for ectomycorrhizal synthesis of Cenococcum geophilum Fr. with Cathaya argyrophylla Chun et Kuang, an endangered species. Calli initiated from stem segments and adventitious roots differentiated from young seedlings were removed and cocultured with Cenococcum geophilum on a modified Murashige-Skoog medium. Fungal hyphae were visible within intercellular spaces of the callus 4 weeks after inoculation, but definite and well-developed Hartig net structures did not form in the calli 8 weeks after inoculation. The typical ectomycorrhizal structures (i.e. hyphal mantle and intracortical Hartig net) were observed in root segments 8 weeks after inoculation. This is the first report of aseptic ectomycorrhizal-like formation/infection between root organ/callus of Cathaya argyrophylla and the ectomycorrhizal fungus Cenococcum geophilum. This culture system is useful for further investigation of mycorrhizal synthesis in Cathaya trees.（Author for correspondence. Tel: +86 (0)451 8219 1783; Fax: +86 (0)451 8219 1795; E-mail: lumin-fu@163.com）     

Effects of imidazole-4-carboxamide and 2-azahypoxanthine on the growth and ectomycorrhizal colonization of Pinus densiflora seedlings inoculated with Tricholoma matsutake

Imidazole-4-carboxamide (ICA) and 2-azahypoxanthine (AHX) obtained from Lepista sordida inhibit and promote the growth of herbaceous plants, respectively. In this study, we examined the effects of these compounds on the growth and ectomycorrhizal (EM) colonization of Pinus densiflora seedlings inoculated with Tricholoma matsutake that forms EM associations with pines. The EM colonization by T. matsutake was observed on the root systems of P. densiflora seedlings treated with and without ICA and AHX. The growth of both non-EM and EM P. densiflora seedlings was inhibited by ICA, regardless of the EM colonization. In contrast, AHX promoted the growth of non-EM P. densiflora seedlings, but not of EM seedlings, suggesting that EM colonization interferes with the effect of AHX on P. densiflora growth.     

In vitro plantlet formation from adventitious buds on juvenile seedlings of Hinoki cypress (Chamaecyparis obtusa)

Adventitious buds were induced on one-week-old juvenile seedlings of Hinoki cypress on Campbell and Durzan's basic medium containing 10 µM (2.25 mg/l) benzyladenine (BA) and 0.027 µM (0.005 mg/l) naphthaleneacetic acid (NAA) and they elongated into shoots on the same medium lacking benzyladenine. About 70% of shoots could be rooted on the same basic medium containing 14.8 µM (3 mg/l) indole-3-butyric acid (IBA), 0.54 µM (0.1 mg/l) naphthaleneacetic acid and 2.66 µM (1 mg/l) riboflavin. These plantlets were potted in perlite. They were micropropagated about 100 times per seedling within 18 months.Present address (until 3rd, October 1986): Katsuaki Ishii, CSIRO Division of Forest Research, PO Box 4008, Canberra, A.C.T. 2600 Australia     

草莓试管苗分化培养基优化的研究

本研究采用三种培养基（A3、F、MS）进行草莓分化对比试验．结果表明我们研制的A3基对草莓试管苗分化有明显促进作用．连续培养11周,A3的草莓苗总分化率是F基的1．25倍,是MS基的1．51倍。而且,A3基成本低于其它两基。A3、F和MS无机盐含量分虽为：1782．58mg／L、2868．33mg／L和4633．33mg／L。无机盐种类的合理配比及其低浓度有利于草莓试管的分化培养。     

In vitro ectomycorrhizal formation in Picea glehnii seedlings

 Twenty isolates of ectomycorrhizal fungi – 3 from Picea glehnii, 12 from other coniferous trees, and 5 from decidous trees – were tested for the ability to form mycorrhizae with P. glehnii, using an in vitro synthesis technique. Macroscopically, mycorrhizal formation was observed 3 months after inoculation, when the lateral roots began to grow. Mycelial growth was observed in all inoculated treatments, generally around and along the roots. Six months after inoculation, seedlings were harvested and the mycorrhizae were observed microscopically. Fourteen of the 20 isolates formed ectomycorrhizae with a dense sheath and a deep Hartig net; 1 formed ectendomycorrhizae with a rudimentary mantle, a well-developed Hartig net and intracellular hyphae; 3 formed pseudomycorrhizae with a mantle but without the Hartig net; and only 2 of the fungi tested, Chalciporus pipeparatus 5/92 and Lyophyllum sp. 61/92, did not form mycorrhizae at all. P. glehnii was a good host species since it had low specificity to ectomycorrhizal fungi isolated from trees other than P. glehnii. Accepted: 6 May 1996     

Plantlet formation from embryonic tissue of chestnut grown in vitro

Development of axillary shoots was induced when isolated embryonic axes of chestnut (Castanea sativa Mill.) were cultured on a defined medium containing 6-benzyl-aminopurine (BAP). The optimal concentrations of BAP were determined for development of axillary shoots from both embryonic axes and subcultured shoots. After shoot multiplication a great number of shoots have been maintained sequentially without significant change in the proliferative rate for one year. Limited rooting has been obtained with excised shoots. Indole-3-butyric acid (IBA) at 2 mg/l was used to induce root primordia. After 8 days of treatment the plantlets were transferred to an auxin-free medium for root development.     

An in vitro method for establishing mycorrhizae on coniferous tree seedlings

Summary A method for in vitro synthesis of mycorrhizae on coniferous tree seedlings is described. Tree seedlings (Larix decidua Mill., Picea abies (L.) Karst, and Pinus sylvestris L.) and fungi Amanita muscaria (L. ex Fr.) Hooker, Piloderma croceum Erikss. et Hjorst., Pisolithus tinctorius (Pers.) Coker et Couch, and Suillus grevillei (Klotzsch) Singer were maintained under sterile conditions in petri dishes. Typical ectomycorrhizae were established within 2–3 weeks after inoculation and within 2 months after germination of seedlings. Eventually a high percentage of mycorrhizal root tips was obtained.     

Factors affecting callus and shoot formation from in vitro cultures of Lens culinaris Medik.

The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these calli. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg l^–1 of BA and 0.186 mg l^–1 NAA+2.25 mg l^–1 BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.     

束状刺盘孢的体外培养条件研究

目的对束状刺盘孢体外培养,观察形态特征。方法将束状刺盘孢接种在马铃薯葡萄糖琼脂培养基(PDA)中分别置于4℃、28℃、35℃和37℃下培养2周,观察生长情况;选取生长最好的菌株分别接种在沙堡弱培养基(SDA)、PDA、察氏培养基(CPA)、胡萝卜琼脂培养基(CDA)和玉米粉培养基(CMA)中,同一温度下培养2周,观察菌落形态及镜下形态。结果菌株在28℃条件下生长最快、菌落发育饱满、产生灰色色素;菌株在五种培养基中生长快慢依次是PDA>CDA>CMA>SDA>CPA,菌落在PDA、CDA和CMA中呈鼠灰色,SDA中呈白色和棕色,CPA中呈白色和鼠灰色,SDA和CPA中未见分生孢子和刚毛产生。结论在PDA、CDA和CMA中28℃条件下,较适合束状刺盘孢生长。     

In vitro bone formation on coral granules

Summary We investigated the ability of fetal rat bone cells isolated after collagenase digestion to differentiate in vitro and to produce a mineralized matrix on coral granules. Scanning electron microscopy examination of the surface of the seeded coral granules revealed that cells attached, spread, and proliferated on the material surface. Bone nodule formation was studied in this in vitro system by direct examination under an inverted phase contrast microscope. The initial event observed was the appearance of cells with phosphatase alkaline activity arranged in several layers and forming a three-dimensional organization around the coral particles. By Day 7, nodule formation began and a refringent material appeared and extended to the background cells during the following days. By Day 15, some coral granules were embedded in a mineralized matrix. Histologic results demonstrated the formation of a mineralized tissue with the appearance of woven bone. This work was supported by the University of Paris VII and the Dental Association of Garancière. This work is a part of Doctorat d'Université that J. M. Sautier will be submitting to the University of Paris VII.     

小鼠2,4,8—细胞胚胎卵裂球体外培养的研究

以ＣＺＢ为基础培养液,培养小鼠２、４、８－细胞胚胎的卵裂球,研究葡萄糖、牛磺酸和胰岛素对１／２卵裂球体外发育的影响及１／４、１／８和２／８卵裂球的体外发育规律。２－细胞胚胎卵裂球在ＣＺＢ中和在添加牛磺酸的ＣＺＢ中培养,其囊胚发育率（分别为９２％、８９％）无显差异（Ｐ＞０．０５）。胰岛素在少量葡萄糖存在的情况下,不影响卵裂球的囊胚发育率;在无葡萄糖时,卵裂球的囊胚发育率（３８％）显降低。牛磺酸在     

In situ and in vitro colonization of Cathaya argyrophylla (Pinaceae) by ectomycorrhizal fungi

Cathaya argyrophylla, a critically endangered conifer, is found to grow at four isolated areas located in subtropical mountains of China. To examine the involvement and usefulness of mycorrhizas for sustaining the population of this tree, we compared the root system, morphology, and structure of mycorrhizal roots of C. argyrophylla, which were collected from a natural stand and an artificial stand, each grown at a different location. More mycorrhizal roots were found for trees from an artificial stand. The presence of extramatrical mycelium, mantle, and Hartig net revealed that C. argyrophylla formed an ectomycorrhizal association in both sampling sites. Starch granules were found in mycorrhizal roots collected only from a natural stand. The aseptic synthesis of C. argyrophylla and Cenococcum geophilum was established for the first time in vitro. Typical ectomycorrhizas formed on seedlings on RM medium containing 0.1 g/l glucose, 5 weeks after inoculation. By light microscopy, the synthesized mycorrhizas showed a thin mantle from which emanated extramatrical hyphae and highly branched Hartig net. A simple, rapid, and convenient mycorrhiza synthesis system was developed, which facilitates further studies on ectomycorrhizal development of C. argyrophylla.