Protoplast regeneration and monokaryotation characteristics of Cyclocybe aegerita

Abstract:

[Background] Genetic breeding is the key guaranteeing the sustainable development of the Cyclocybe aegerita industry. The preparation of protoplasts and monokaryons can provide technical support for the genetic breeding of C.aegerita. [Objective] To obtain the regeneration characteristics, monokaryotation characteristics, and mating types of the protoplasts for the cross breeding, fusion breeding, mutagenesis breeding, genetic transformation, and functional gene mining of C.aegerita. [Methods] The mycelia of C.aegeritaAa11 were directly treated with mannitol solution and lysozyme solution to prepare the protoplasts which were then isolated and regenerated. The changes of colony morphology were observed during the hybridization of protoplasts derived from mononuclear mycelia. [Results] When 7 blocks were inoculated for enzymatic hydrolysis at 33–34 ℃ for 60–80 min, the suspension reached a concentration of 10⁷ protoplasts/mL. The protoplasts of C.aegeritacould not form regenerative colonies until 7 days after the coating and showed a regeneration rate of 0.71% and a monokaryotation rate of 41.1% on the regeneration medium. The regenerated dikaryon and monokaryon strains presented a time difference in the formation of colonies. From day 7, all the regenerated colonies in 3 consecutive days were heterokaryonic strains, and mononuclear colonies began to appear successively since day 4. Almost all of the colonies in the following days were mononuclear strains. A total of 290 monokaryons were obtained in this study and classified into two parental mating types A1B1 and A2B2 (A1B1:A2B2 ratio of 138:152). The monokaryons of both mating types showed vigorous white aerial hyphae, and the monokaryon growth of A1B1 was faster than that of A2B2. A dividing line, bending towards the parent colony with a slower expansion rate, appeared in the contact area of the parent colonies, with the mycelia significantly sparser than the surrounding mycelia. [Conclusion] The morphological characteristics of the two monokaryon strains and their hybridization provide a basis for the selection of parents in the cross breeding of C.aegerita.