Formation and regeneration of protoplasts from the ectomycorrhizal basidiomycete Laccaria bicolor

1986 ◽  
Vol 64 (6) ◽  
pp. 1224-1226 ◽  
Author(s):  
Bradley R. Kropp ◽  
J. A. Fortin
Keyword(s):  
Pinus Banksiana ◽  
Enzyme Preparation ◽  
Lytic Enzyme ◽  
Laccaria Bicolor ◽  
Dikaryotic Mycelium ◽  
Osmotic Stabilizer

Protoplasts were released from dikaryotic mycelium of the ectomycorrhizal basidiomycete Laccaria bicolor using the lytic enzyme preparation NovoZyme 234. Protoplast release depended strongly on mycelium age, osmotic stabilizer, and temperature. The protoplasts could regenerate to form both monokaryotic and dikaryotic cultures capable of forming normal ectomycorrhizae with Pinus banksiana.

Differences in the colonization of Pinus banksiana roots by sib-monokaryotic and dikaryotic strains of ectomycorrhizal Laccaria bicolor

1989 ◽  
Vol 67 (6) ◽  
pp. 1717-1726 ◽  
Author(s):  
Ken K. Y. Wong ◽  
Yves Piché ◽  
Diane Montpetit ◽  
Bradley R. Kropp
Keyword(s):  
Pinus Banksiana ◽  
Intraspecific Variability ◽  
Root Cell ◽  
Laccaria Bicolor ◽  
Cortical Cells ◽  
Aseptic Culture ◽  
Hartig Net ◽  
Cell Layers ◽  
The Mean ◽  
Root Surfaces

First-order laterals of Pinus banksiana seedlings were inoculated with variant strains of ectomycorrhizal Laccaria bicolor in an aseptic culture system. Macroscopic observations of 10 fungal strains indicated that 6 are mycorrhizal and 4 are apparently nonmycorrhizal. Furthermore, light microscopic examinations revealed significant intraspecific variation in mycorrhizal structures. The mean mantle thickness, mean mantle density, and mean Hartig net penetration of the six mycorrhizal strains ranged from 2.5 to 13.4 hyphae, 278 to 411 hyphae/mm and 2 to 2.8 root cell layers, respectively. Three of these strains formed fewer macroscopically observable mycorrhizae and developed significantly thinner mantles but their Hartig nets usually separated cortical cells more extensively. Three of the four apparently nonmycorrhizal strains showed infrequent and poor Hartig net development (mean penetration of 0.3 to 0.8 root cell layer), poor surface colonization, and no mantle development. These three strains were better able to colonize long roots. Only one strain could be considered truly nonmycorrhizal because it only colonized root surfaces poorly and never showed mantle or Hartig net formation. The observed intraspecific variability raises questions concerning the determinants of mycorrhiza development and structure.

A Petri dish technique for the aseptic synthesis of ectomycorrhizae

1989 ◽  
Vol 67 (6) ◽  
pp. 1713-1716 ◽  
Author(s):  
Ken K. Y. Wong ◽  
J. André Fortin
Keyword(s):  
Pinus Banksiana ◽  
Petri Dish ◽  
Microbial Interactions ◽  
Laccaria Bicolor ◽  
Larix Laricina ◽  
Hebeloma Cylindrosporum ◽  
First Order ◽  
Whole Plants ◽  
Improved Technique ◽  
Physiological And Biochemical

An improved technique was developed for the aseptic synthesis of ectomycorrhizae in Petri dishes. Within 2 weeks after inoculation of a first-order lateral, ectomycorrhizae were formed on Larix laricina and Pinus banksiana with Hebeloma cylindrosporum, Laccaria bicolor, and Pisolithus tinctorius. The technique should be useful for addressing physiological and biochemical questions concerning microbial interactions with roots of whole plants.

Protoplast release from fungi capable of steroid transformation

1984 ◽  
Vol 30 (1) ◽  
pp. 57-62 ◽  
Author(s):  
J. Długoński ◽  
L. Sedlaczek ◽  
A. Jaworski
Keyword(s):  
Cell Wall ◽  
Enzyme Preparation ◽  
Trichoderma Viride ◽  
Transformation Product ◽  
Lytic Enzyme ◽  
Cunninghamella Elegans ◽  
Steroid Hydroxylation ◽  
Steroid Transformation ◽  
No Inhibition ◽  
Protoplast Suspension

Protoplasts were obtained from Hyphoderma roseum (Fries) and Cunninghamella elegans (Lendner), fungi capable of steroid 11-hydroxylation. The lytic enzyme preparation was derived from Trichoderma viride CBS 354-33. Homogeneous protoplast suspension, free of mycelial debris and cell wall fragments, transformed cortexolone and 6α-fluorocortexolone-16,17-acetonide to the same products as the intact mycelium of the microorganisms. Liberation of protoplasts and their stabilizaiton during steroid transformation was the most effective in 0.8 M MgSO4; still, this compound impaired steroid hydroxylation. Consequently, the concentration of the transformation product formed was nearly the same as in sucrose, mannitol, and sorbitol, compounds which caused no inhibition but which were less effective stabilizers.

Release of protoplasts from the galactose oxidase producing mold, Dactylium dendroides

1983 ◽  
Vol 29 (7) ◽  
pp. 763-766 ◽  
Author(s):  
Déa Amaral ◽  
Doroty Kubicki ◽  
Hector F. Terenzi
Keyword(s):  
Growth Medium ◽  
Phosphate Buffer ◽  
Normal Growth ◽  
Galactose Oxidase ◽  
Lytic Enzyme ◽  
Considerable Importance ◽  
Digestive Juice ◽  
Osmotic Stabilizer ◽  
Dactylium Dendroides

Osmotically sensitive protoplasts were released from the mycelium of Dactylium dendroides, using Megalobolinus paranaguensis digestive juice as a lytic enzyme. The conditions for obtaining stable protoplasts were determined. The maximum number of protoplasts was obtained from 15-h growing mycelium, using MgSO4 as osmotic stabilizer, in the presence of 0.015 M Sorensen phosphate buffer, pH 5.6. MgSO4 proved to be of considerable importance in the release of protoplasts in this fungus. Regeneration of the protoplasts was demonstrated in normal growth medium supplemented with 0.8 M mannitol and 2.5% agar.

"PROTOPLASTS" FROM PYTHIUM SP. PRL 2142

1967 ◽  
Vol 13 (12) ◽  
pp. 1701-1704 ◽  
Author(s):  
J. H. Sietsma ◽  
D. E. Eveleigh ◽  
R. H. Haskins ◽  
J. F. T. Spencer
Keyword(s):  
Lytic Enzyme ◽  
Enzyme Complex ◽  
Enzyme Preparations ◽  
Streptomyces Spp ◽  
Osmotic Stabilizer ◽  
Enzyme Complexes

Enzyme preparations from two Streptomyces spp. released spherical protoplast-like bodies from the mycelium of Pythium sp. PRL 2142. The enzyme complexes were produced by growing the Streptomyces spp. in the presence of autoclaved Pythium mycelium, and concentrated from the culture mixture by precipitation with acetone (2:1, v/v). The protoplasts were released, from both intercalary and terminal locations on the hyphae, during incubation with the lytic enzyme complex for 24 hours using 0.8 M MgSO4 as an osmotic stabilizer.

Variable loss of ectomycorrhizal ability in monokaryotic and dikaryotic cultures of Laccaria bicolor

1987 ◽  
Vol 65 (3) ◽  
pp. 500-504 ◽  
Author(s):  
Bradley R. Kropp ◽  
Brenda J. McAfee ◽  
J. André Fortin
Keyword(s):  
Mating Type ◽  
Pinus Banksiana ◽  
Laccaria Bicolor ◽  
Relative Ability

The relative ability of sibling monokaryotic and reconstituted dikaryotic cultures of Laccaria bicolor (Maire) Orton to colonize short roots of Pinus banksiana Lamb. was studied using two different methods at 4, 8, and 12 months after isolation. Monokaryotic isolates varied in their ability to form ectomycorrhizae. Some lost the ability to colonize rootlets. Dikaryons formed soon after isolation from compatible mating type pairs were vigorous colonizers. A dikaryon formed after 12 months by pairing two competitive monokaryons formed abundant mycorrhizae. However, dikaryons formed after 12 months by using less competitive monokaryons showed a sharply reduced symbiotic ability. The isolation of one apparently nonmycorrhizal monokaryon is reported.

Release and regeneration of protoplasts from the fungus Trichothecium roseum

2003 ◽  
Vol 49 (4) ◽  
pp. 263-268 ◽  
Author(s):  
N Balasubramanian ◽  
G Annie Juliet ◽  
P Srikalaivani ◽  
D Lalithakumari
Keyword(s):  
Yeast Extract ◽  
Parent Strain ◽  
Protoplast Isolation ◽  
Lytic Enzyme ◽  
Complete Medium ◽  
Lytic Enzymes ◽  
Trichothecium Roseum ◽  
Osmotic Stabilizer ◽  
Enzyme Combination ◽  
Yeast Extract Agar

A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 × 104 protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose – yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.Key words: Trichothecium roseum, protoplast, isolation, regeneration, lytic enzymes, osmotic stabilizers.

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