Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Mushroom
Reexamination Certificate
1998-01-05
2003-02-18
Campbell, Bruce R. (Department: 1661)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Mushroom
C800S295000, C047S001100
Reexamination Certificate
active
06521817
ABSTRACT:
The present invention relates to novel hybrid strains of
Agaricus bisporus
whose fructifications have a mostly tetrasporous and/or trisporous nature under standard culture conditions and allow homocaryons to be obtained in large amounts.
The invention also relates to a process for the production of such hybrid strains and/or to the use of these hybrid strains in a process for obtaining and selecting improved strains of
Agaricus bisporus.
Furthermore, the invention relates to the use of such hybrids which have mostly tetrasporous and/or trisporous fructifications for obtaining considerable amounts of homocaryons and/or in a process for obtaining a bisporus Agaricus strain which has been improved by carrying out a cross (crosses) and by selection.
If the present application uses the term “strain” without being more specific, this is to be understood as meaning a strain of
Agaricus bisporus
or a strain which is interfertile with
Agaricus bisporus.
Moreover, the term “tetrasporous”, without being more specific, can denote, for example, “mostly tetrasporous and/or trisporous, and to a minor extent bisporous”, in particular when it is clear that the intended aim is to be able to have a large number of homocaryons available. The term “having mostly tetrasporous basidian” can, for analogous reasons, also denote “whose spores are mostly homocaryotic”. The term “homocaryotic spore” here indicates spores whose germination provides homocaryotic mycelia (or homocaryons).
It is known that the species of the genus Agaricus have basidia which are usually tetrasporous. The cultivated mushroom is a notable exception, having mostly bisporous basidia (generally in an amount of at least 90%). From this results the name of this species:
Agaricus bisporus.
The spores of
Agaricus bisporus
normally contain two non-fraternal nuclei generated by meiosis, and their germination into a monosporous culture gives fruiting heterocaryotic mycelia, that is to say they are capable of fruit formation without confrontation with a compatible mycelium.
Agaricus bisporus
is therefore a secondary homothallic bisporous species, according to the definition of J. R. RAPER, “Genetics of Sexuality in Higher Fungi”, The Remaid Press Cy, New York, 1966. Its sexual type is bipolar, which can be expressed by the fact that each of the mycelia from one of the four spores of one of the rare tetrasporous basidia is compatible with two of the mycelia from the other three spores.
In view of its homothallic nature, it is difficult to obtain large amounts of homocaryons of
Agaricus bisporus,
which would be necessary for carrying out selection strategies by crossing.
This is partly why the genetic variability between the strains which are traditionally grown is, in fact, restricted to six classes of genotypes, two of these classes having the characteristic white which originates from mutants which appeared during the cultivation, one in 1926 and the other in 1960. A seventh class termed “hybrid class” results from a cross which was carried out between the two white strains.
Moreover, there are wild-type strains of
Agaricus bisporus,
but these strains have a dominant brown characteristic which makes it difficult for them to be used in the generation of hybrids.
In connection with everything that has been said in this matter, for example, G. Fritsche and A. S. M. Sonnenberg, “The cultivation of Mushrooms” Van Griensven Ed. (1988), pages 101-123, can be cited.
The existing methods for obtaining homocaryons of
Agaricus bisporus
are difficult to carry out and, above all, only give rise to small amounts of different homocaryons. These methods consist either in germinating the spores and isolating and identifying mycelia from spores of the rare tetrasporous basidia, or in isolating the spores from tetrasporous basidia by means of micro-manipulation, or else in isolating protoplasts by means of enzymatic lysis of the wall of multinucleate heterocaryotic cells, which constitute the mycelium; see, for example, G. Fritsche and A. S. M. Sonnenberg, the paper cited above.
It is known that a range of methods can be used to identify homocaryotic mycelia and to distinguish them from heterocaryotic mycelia, for example:
observation of the mycelial growth, which is generally less vigorous in the case of the homocaryotic mycelia;
experiments in which the homocaryotic mycelia are confronted with homocaryons having different incompatibility alleles;
experiments on the formation of fruiting bodies;
the use of one or, preferably, more biochemical markers, in particular enzymatic markers, especially alloenzymes (May and Royse, Experimental Mycology, Vol. 6, pages 283-292, 1982;
or else the use of one or more molecular markers (for example RFLP or RAPD markers); see, for example, “Genetics and Breeding of Agaricus”, Proceedings of the First International Seminar on Mushroom Science, Horst (Netherlands), May 14-17, 1991, L. J. L. D. Van Griensven Ed, PUDOC Wageningen 1991, in particular pages 62-72 (P. A. Horgen et al.) and pages 73-77 (R. S. Khush et al.).
The biochemical and molecular markers can be used in two ways: either it is known that the mother strain is heterozygous, and for example the loss of heterozygosis in the test mycelium shows that it must be homocaryotic, or else a mycelium M which is presumed to be homocaryotic is confronted with an intercompatible homocaryon carrying an allele which differs from the marker being studied, and if the resulting mycelium is heteroallelic, the mycelium M was indeed homocaryotic, and in this latter case the use of a single marker suffices to draw conclusions.
It has now been found that there are wild-type strains of Agaricus which are bisporous to a very minor extent, of which for example at least 80% of the basidia have more than two spores (in particular four spores), which are interfertile with the wild-type strains and the commercial strains of
Agaricus bisporus,
and whose hybrids with
Agaricus bisporus
are mostly tetrasporous and/or trisporous without any particular measures to this end being taken. In other words, this trait of being tetrasporous and to a minor extent bisporous is dominant in such a hybridization. Moreover, said hybrids themselves are interfertile with
Agaricus bisporus.
Such wild-type strains can be found in particular in California in the Sonoran desert. One of these strains is supplied under the no. ARP 61 by the Agaricus Recovery Program (abbreviated: ARP) Post Office Box 461, Worthington Pa. 16262, USA.
Other analogous strains which are bisporous to a minor extent, such as ARP 15 (also termed “JB2” and deposited at the ATCC under the no. 76072) and possibly certain strains selected from amongst strains ARP 116 to 167 can also be obtained from the ARP.
Because of the fact that the tetrasporous phenotype of these wild-type strains can surprisingly be dominantly transmitted to hybridization products, it is possible to use those wild-type strains whose fructifications have mostly homocaryotic spores to carry out crosses with strains of
Agaricus bisporus
which give rise to hybrids whose tetrasporous and/or trisporous nature (the majority of the spores thus being homocaryotic) allows homocaryons to be obtained for the first time in considerable quantities, of which it is known that they are required for carrying out subsequent hybridization crosses with a view to obtaining improved hybrid varieties by selection.
It must be remembered that the rare known cases of strains in which a sizeable number of basidia have more than two spores were the result of specific culture conditions (environment) and not of a trait which can be transmitted genetically; see, for example, J. Pelham, Mushroom Science 7, pages 49-64 (1965) and S. F. Song et al., Mushroom Science 8, 295-303 (1972). It should be noted here that in the tetrasporous strains used and/or obtained according to the present invention, the tetrasporous nature can be transmitted and is observed under standard culture conditions, which can be, for example, the following: compost on the basis of horse manure and straw
Billette Christophe
Callac Philippe
Imbernon Micheline
C.T.C. Centre Technique du Champignon et Institut
Campbell Bruce R.
Oliff & Berridg,e PLC
Para Annette H.
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