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Conservation et instabilité des souches de champignons filamenteux Impact de la cryoconservation
Abstract
Présentation critique des diverses méthodes de conservation des souches de champignons filamenteux et situation de la cryoconservation dans ce contexte. Les traitements de conservation (lyophilisation et cryoconservation) peuvent provoquer des destructions d'ampleurs variables selon l'âge et la vigueur des souches, destructions qui menacent la survie globale dès qu'elles ne sont plus masquées par l'effet de masse: le conditionnement préalable des thalles est donc un élément déterminant de la survie.
Basidiomycetes are used in industrial processes, in basic or applied research, teaching, systematic and biodiversity studies. Efficient work with basidiomycete cultures requires their reliable source, which is ensured by their safe long-term storage. Repeated subculturing, frequently used for the preservation, is time-consuming, prone to contamination, and does not prevent genetic and physiological changes during long-term maintenance. Various storage methods have been developed in order to eliminate these disadvantages. Besides lyophilization (unsuitable for the majority of basidiomycetes), cryopreservation at low temperatures seems to be a very efficient way to attain this goal. Besides survival, another requirement for successful maintenance of fungal strains is the ability to preserve their features unchanged. An ideal method has not been created so far. Therefore it is highly desirable to develop new or improve the current preservation methods, combining advantages and eliminate disadvantages of individual techniques. Many reviews on preservation of microorganisms including basidiomycetes have been published, but the progress in the field requires an update. Although herbaria specimens of fungi (and of basidiomycetes in particular) are very important for taxonomic and especially typological studies, this review is limited to live fungal cultures.
One hundred and two fungal strains remained viable by cryogenic preservation for nine years. Two strains survived for only six years. Of the two suspending media used, dimethyl sulfoxide was significantly better than glycerol as a cryoprotectant for eight strains. During storage, seven viability checks were performed. Variability was examined in relation to storage time, frozen-thawed cultures vs. first subcultures, mycelial plugs (discs), and vials. Variation in radial growth of colonies was a function of innate properties of an organism, and there was no evidence that variation was influenced by cryogenic conditions (freezing and storage). The proposed method of preservation was demonstrated to be suitable for long- term conservation of these strains and is presumably applicable to other mycelial cultures.
the early 1900's, before the introduction of the principles of microbial genetics prerequisite to a solution of the problem. There is the added difficulty of the lack of any recent critical attempt at a reexamination of these early findings. It is, consequently, the purpose here to correlate these results with the more recent work of pleomorphism, including my own observations, and to see whether such principles as mutation, selection, and norm of reaction can be used in a reexamination of the phenomenon. Since my experience has been limited to a study of one species, Trichophyton mnentagrophytes, this paper will deal specifically with pleomorphism in this species and in general terms with pleomorphism in the dermatophytes as a whole. The original biological material in this study was one strain of T. mentagrophytes, the N form, first described by Robbins (1945, 1949) and kept in culture, apparently unchanged, up to the present time. It will be referred to here as the N, normal, or conidial form, to distinguish it from the pleomorphic variants derived from it. The nutrient medium used, a thiamine-neopeptone agar (McVeigh and Cambell,
Résumé
Les cultures de tissus et de cellules subissent au cours de leur développement in vitro des variations plus ou moins importantes. La variabilité morphogénétique chez les organes et les plantes issus de ces cultures est fonction de l'importance de ce phénomène et, parfois même, de l'état initial des cellules. Elle touche généralement la morphologie, le port de la plante, la forme des feuilles, des inflorescences, des fleurs et se répercute sur la fertilité, mais on rencontre aussi d'autres anomalies. Elle prend très souvent de l'ampleur avec l'âge des souches tissulaires et peut conduire à la production de véritables monstres. Au sein des exemples choisis pour l'illustrer, le Tabac a été pris comme exemple type. Des cas particuliers de variations sont également décrits. L'article se termine par quelques réflexions sur cette variabilité et les espoirs qu'elle suscite.
De nombreux résultats expérimentaux, issus de diverses expériences de culture in vitro comportant soit des tissus somatiques, soit des gamétophytes, soit des cellules isolées, ou des protoplastes, obligent à postuler d'une part un système souple d'expression allélique, le linkat, d'autre part un réseau de signaux moléculaires regroupés dans un compartiment épigénique. Une tentative de définition de ces structures hypothétiques est présentée ainsi que quelques sites ou systèmes biologiques qui pourraient être impliqués.
The eventual causes of the emergence of heritable phenotypic variations in plant cell cultures are discussed.In a first part, the mechanisms of cell alteration whose nature can be genetic or epigenetic are considered; the ambiguity of the acceptation of these two words is discussed and the criteria used to determine the genetic or epigenetic nature of the variations are reported.The second part deal with the selection of various cellular types inside the cell cultured populations according to the selection pressure induced by environmental conditions. So we are faced with the following problem: either the pre-existence of an in vivo initial cellular heterogeneity, or the induction of this heterogeneity by in vitro culture. The few experimental results on this point induce to think that the plant cell variability is expressed permanently and with the same range in vivo and in vitro, giving rise to new cellular types. Only the pressure of a modified environment could allow or not to the disclosure of the corresponding phenotypic variations.
Résumé
L'objectif de cet article est d'illustrer les différences de modalités du comportement de fragments prélevés sur un végétal, d'une part en relation avec ce qui s'est passé à leur niveau pendant et après leur mise en place dans l'individu, d'autre part en fonction des conditions nouvelles, particulières dans lesquelles se forge in vitro leur nouveau système de corrélations.
A method is described by which over 3000 isolates of microfungi have been successfully frozen and stored in liquid nitrogen for up to 13 years. The majority of the isolates that failed to survive were from the Mastigomycotina. Results of successful and unsuccessful storage of Phytophthora and Pythium species and lower Phycomycetes are presented. Out of 189 isolates of Phytophthora 108 were successful, though out of 58 isolates of Pythium only 27 survived.
The capacity of Pestalozzi annulata B. & C. for mycelial differentiation is controlled by the genome. The transition between the juvenile and the differentiated state is under extra-chromosomal control and is reversible and controlled metabolically.
One hundred and two fungal strains remained viable by cryogenic preservation for nine years. Two strains survived for only six years. Of the two suspending media used, dimethyl sulfoxide was significantly better than glycerol as a cryoprotectant for eight strains. During storage, seven viability checks were performed. Variability was examined in relation to storage time, frozen-thawed cultures vs. first subcultures, mycelial plugs (discs), and vials. Variation in radial growth of colonies was a function of innate properties of an organism, and there was no evidence that variation was influenced by cryogenic conditions (freezing and storage). The proposed method of preservation was demonstrated to be suitable for long-term conservation of these strains and is presumably applicable to other mycelial cultures.
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Conidiated slope cultures of derivative of Penicillium chrysogenum Wis 54-1255 were stored at -196 or +4 C for a period of 3.5 years. After this time, the viability fell to 68% in the former case and to 4% in the latter. At the end of the experiment, 65 single conidial isolates from each series were tested for penicillin yield. Among those from conidia stored at -196 C, the spread of penicillin yields did not differ markedly from that of 65 single conidial isolates made as controls prior to storage. However, 18% of those from conidia stored at +4 C formed a subpopulation with substantially lower penicillin titers than those of control isolates. Storage at -196 C may reduce or prevent a possible source of penicillin yield decay, namely, the selection of spontaneous mutants of low titer present in small numbers in the original culture and selected, as viability decreased, by virtue of their increased longevity relative to that of the parental culture.
Genetic and transcriptional maps have been constructed for the mitochondrial genome of the Ascomycete Podospora anserina. These data have been plotted on the restriction maps for Sal I, Xho I, Bam HI, Eco RI, BgI II and Hae III. We have characterized and cloned a new and unique senescent mitochondrial DNA (beta-event senDNA) and have organized all of the recognized senDNAs on the genomic maps. We make the observation that all of the known and characterized senDNAs are derived from specific genes or gene regions of the young mitochondrial genome. We have unambiguously assigned the alpha-event senDNA (the 2.6 kb monomer) to the oxi3 gene locus and the beta-event senDNA to the oxi2 gene locus.
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