Methods for production of sporeless
Methods of producing hybrid Agaricus bisporus mushrooms strains derived or descended from at least one wild mushroom strain and having the specified traits of either greatly diminished sporulation or an absence of sporulation, and of obtaining postmeiotic offspring of nonsporlating basidiomycete fungi including Agaricus bisporus , are disclosed.
1. A method for producing a hybrid mushroom strain of Agaricus bisporus and determining that an Agaricus bisporus culture carries a genetic determinant for at least one sporeless trait selected from the group consisting of non-sporulation, reduced sporulation, incomplete development of spores, incomplete release of spores, and paler lamellae, the method comprising:
placing a first culture of Agaricus bisporus and a second culture of Agaricus bisporus in close proximity on a suitable sterile culture medium, wherein said step of placing enables fusion via anastomosis and plasmogamy between the first culture of Agaricus bisporus and the second culture of Agaricus bisporus , wherein the first culture is a homokaryoni an aneuploid, or a karyotypically indeterminate culture that has been identified to be germ plasm capable of transmitting to the offspring at least one sporeless trait, to produce the hybrid mushroom strain of Agaricus bisporus ;
wherein when the hybrid strain obtained from said fusion has an expressed trait for a sporeless phenotype, the second culture is determined to carry a genetic determinant for at least one sporeless trait; and
wherein said method does not use any genetic marker or MAS technique to mark alleles of any gene hypothetically associated with the production of a sporeless phenotype in mushrooms of Agaricus bisporus.
2. The method according to claim 1 , wherein a new hybrid Agaricus mushroom strain is produced, the method comprising:
selecting the culture that has been determined to carry the genetic determinant for the at least one sporeless trait; and
carrying out hybridization between a third culture of Agaricus bisporus and the selected culture, said step of carrying out hybridization including a step of placing the two cultures in close proximity on a suitable sterile culture medium, wherein said step enables fusion via anastomosis and plasmogamy between the two cultures, thereby producing the new hybrid Agaricus mushroom strain, and wherein the selected culture is selected from the group consisting of homokaryons, aneuploidy, or karyotypically indeterminate cultures.
3. A method for obtaining viable recombinant postmeiotic homokaryotic or heterokaryotic offspring from a sporeless strain of a basidiomycete fungus comprising:
selecting basidiomata from a sporeless strain of a basidiomycete fungus at a stage of development in which meiosis has occurred in some basidia; axenically excising lamellae from said basidiomata;
applying a technique for repartitioning of contents of postmeiotic cells of said lamellae into vegetatively growing mycelial cultures, said technique being selected from
(a) enzymatically making and regenerating protoplasts from the excised lamellae, or
(b) mechanically reducing the post-meiotic cells by performing (i) microsurgery or (ii) laser surgery to sever hyphal tips or fragment mycelia;
wherein the postmeotic cells are basidia, or are vegetative hyphae developed from basidia among explants of lamellar tissue.