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Review
Long-term
cryopreservation
of
basidiomycetes
Giani
Andrea
Linde,
Alana
Luciani,
Ana
Daniela
Lopes
∗,
Juliana
Silveira
do
Valle,
Nelson
Barros
Colauto
UniversidadeParanaense,ProgramadePós-graduac¸ãoemBiotecnologiaAplicadaàAgriculturadaUniversidadeParanaense, Umuarama,PR,Brazil
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t
i
c
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e
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n
f
o
Articlehistory:Received30December2016 Accepted10August2017 Availableonline18October2017 AssociateEditor:AndreRodrigues
Keywords: Preservation Substrate Fungi Mycelialviability Cryoprotectant
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Basidiomyceteshaveseveralbiotechnologicalandindustrialapplicationssuchasenzyme production,bioremediation,pharmaceuticalandfunctionalfoodproduction.Duetoclimatic features,thepreservationofseveralbasidiomycetesisthreatened,andtoguaranteethe preservationofthisgeneticresource,thedevelopmentoflong-termpreservationtechniques isnecessaryoncethereisnouniversalprotocolforthecryopreservationofbasidiomycetes. Cryopreservationisatechniqueinwhichmicroorganismsaresubmittedtoultralow tem-peratures.Therefore,thisstudyaimedtocollectinformationonthemainconditionsfor long-termcryopreservationofbasidiomycetesinthelast20years.Scientificarticleson cryo-preservationofbasidiomycetespublishedfrom1997to2016,wereresearched,andonlythe studiesontwointervalsofcryopreservationwereconsidered:from1to2yearsandfor longerthan2years.Theanalyzedconditionsofbasidiomycetecryopreservationwere:most studiedgenera,cryopreservationtemperature,substrate,cryoprotectant(andpreservation substrate),cryopreservationperiod,thawing temperatureand cultivationmedium after thawing,physiologicalandgeneticstabilityofbasidiomycetesafterthawingin cryopreserva-tion.Inthisreview,theviabilityofthemaincryopreservationconditionsofbasidiomycetes studiedinthelast20yearsarepresentedanddiscussed.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Basidiomycetesareutilizedtoconvertagriculturaland agroin-dustrialresiduesintobiomass,enzymesorbioremediation.1–3 Laccase production by these fungi has been applied to blanching/decoloringandbiopulping4–6 andtobiodegradate ofxenobioticpollutants.7,8 Furthermore,thespentsubstrate
∗ Correspondingauthor.
E-mail:anadanielalopes@unipar.br(A.D.Lopes).
frommushroomproductionhasbeenusedasahighly valu-ableorganicfertilizer,developingtheeconomyofecological recycling.9
Basidiomycetesarealsofundamentaltothedevelopment of pharmaceutical and functional products10 species such asGrifolafrondosa(Dicks.)Gray,Ganodermalucidum(Curtis)P. Karst,Lentinulaedodes(Berk.)Pegler,Pleurotusostreatus(Jacq.ex Fr.)P.Kumm.,Volvariellavolvacea(BulliardexFries)Singer11,12
https://doi.org/10.1016/j.bjm.2017.08.004
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
andAgaricussubrufescensPeck(AgaricusblazeiMurrill; Agari-cusbrasiliensisWasseretal.)13,14amongothers.Thesespecies arecharacterizedbytheircontentof-dglucans, proteogly-cans,triterpenes,phenoliccompounds, amongothers,with immune-stimulating, antitumoral, hypoglycemic, antifun-gal,antiviral,antibacterial,anti-inflammatory,antiallergenic, antioxidant,andhypocholesterolemicactivity.11,12,15–17
It is estimated that the Fungi kingdom consists of 1.5 million species, and 70,000 have been described, 14,000 produce known mushroom and 650 have pharmacological properties.18However,only10speciesarecultivatedin com-mercialscale.9Thisimpliesthatanenormousbiodiversityof basidiomyceteshasnot beenexplored yet. Thus, itis nec-essarytopreservethesespecies.However,climaticchanges haveaffected ecological systemschanging the lifecycle of fungi19,20,21whichmaycausealossofgeneticdiversity.The growing industrial importance of fungi and inherentrisks ofclimaticchanges implyvulnerability ofgenetic diversity whichwillbereducedwiththedevelopmentofpreservation techniques.22
Different preservation methods have been utilized for basidiomycetesandthemostversatiletechniqueis cryopres-ervation,inwhich standarddomestic freezerscanbeused havingprovedtobeidealandeconomicaltoolstokeepfrozen culturecollections.23Lyophilizationmaybeoneofthemost widely used technologies to preserve several fungi and is theprimarytechniqueusedinmostgeneralserviceculture collections.23However,thesetechniquesareinappropriatefor mostbasidiomycetes.24
Anotherwidelyusedmethodisperiodicalsubculturebut it takes up more time, space, reagents and is suscepti-ble to contamination, genetic and physiological long-term changes.25–28 Techniquesforfungalpreservationindistilled water, silica,and mineraloil are less effective in the long run.26,27,29Dependingontheutilizedpreservationtechnique, basidiomycetescanreduceproductivity,losemorphological characteristics,andpharmacologicalactivity.30
Severalfactorsaffectcryopreservationofbasidiomycetes suchasspecies,growthphase,substrate,cultivation condi-tions,cryoprotectant,amongothers.31,32Fewmicroorganisms surviveafterfreezing andthawing,32 mainlydueto cryoin-juriescausedtocellmembraneandcellwall.32
Cryoinjuriesarerelatedtotwomainaspects:theformation ofintracellularicecrystals,andthewaterflowoutwardthe cell(dehydration)increasingtheintracellularconcentrationof solutes.32–34Despitethat,cryopreservationatseveralfreezing temperatures35isthemostrecommendedmethodbyseveral microorganismculturecollectionssuchastheAmericanType CultureCollection(ATCC)andtheNationalCollectionofTypes Cultures(NCTC).36,37 Cryopreservationallowsthatmost cul-turesremainstableforalongperiodoftime,andmayreach upto30years,duetothelowoccurrenceofmetabolic activ-ityatthesetemperatures;thatiswhyitisthemostutilized techniquebymostmicrobialbanks.38However,fewstudieson cryopreservationhavebeencarriedoutwithbasidiomycetes. Themainstudiesonfungalcryopreservationsuggestthat thereisnotauniversaltechniqueorprotocolandthateach species has to be studied separately,39 even though there are generalizationsfor thecryopreservation procedures for
basidiomycetes.40 Also,basidiomycete preservationis diffi-cult duetothepredominance ofvegetativemycelia, which are sensitive to environmental variations, and absence of asexualsporesinmanyspecies.25Therefore,becauseofthe importanceofbasidiomycetesinbiotechnologyandtheneed to verify favorable conditions for the cryopreservation of basidiomycetes in the long run, this study aims to collect informationabout the mainconditionsforlong-term cryo-preservationofbasidiomycetesinthelast20years.
Methodology
Thisbibliographicalreviewcollectedscientificarticleson cryo-preservationofbasidiomycetespublishedfrom1997to2016in thedatabankofWebofScience,WorldWideWebandGoogle Scholar.Thesearchtagswerecryopreservation,mushroom, basidiomycete,preservation andfungi.Themycelial viabil-ity was verified aftercryopreservation, and the treatments wheremycelialgrowthwasequalorgreaterthan75%ofthe replications were considered viable.41 Thestudies on cryo-preservationofbasidiomycetesweredividedintotwogroups: one-yeartotwo-yearcryopreservation(shortterm)and cryo-preservationofmorethantwoyears(longterm).Bothgroups weredividedintosubgroupstoevaluategenus,finalfreezing temperatureof−20◦C,from−70to−86◦Cand−196◦C,
sub-strateand preservationsubstrate used incryopreservation, cryoprotectanttypeandconcentration,andcryopreservation period. In thisreview, any substrate that works preserving myceliaincryopreservationwithoutcryoprotectantswas con-sideredapreservationsubstrate.Onlyforthecryopreservation period,thetreatmentsweredividedintocryopreservationof 1–2years,2–3years,3–5years,ormorethan5years.
Ingeneral,short-termcryopreservationperiodsaremore successful regarding mycelial viability recovery. However, shorttimeperiodsarenotsignificanttodifferentiate physi-ologicalandgeneticcharacteristics,buttheyareeffectiveto verifywhenatechniqueisunviable.Mantovanietal.42 cryo-preserved L.edodes at−70◦C forone, two and three years
and verified loss of mycelial viability in most treatments, between the first and second year;however, afterthe sec-ondandthirdyearofcryopreservation,theviabilitywaskept. Colautoetal.41alsoobservedlossofviabilityafterfouryearsof cryopreservationat−70◦Cin8outof12evaluatedtreatments. Nevertheless,Singhetal.43cryopreserveddifferentstrainsof basidiomycetesinliquidnitrogenandobservedviability main-tenanceformoststrains.
According to Nakasone et al.,44 long-term preservation occurs when cultures are preserved for longer than one year using aspecifictechnique. However,forRyan et al.,39 long-termfungalpreservationoccurswhentheculture preser-vationperiodislongerthantwoyears.AccordingtoMantovani etal.,42onlyaftertwoyearsitispossibletoverifydifferences incryopreservation efficiency.Therefore, inthis review,we willconsideronlybasidiomycetecryopreservationof1-2years (shortterm)andofmorethan2years(longterm). Cryopres-ervationstudiesshorterthanayearwerenotconsidereddue tothe insufficienttimetoevaluatethelong-termtechnical efficiency.
Literature
survey
results
Thesearchforcryopreservationarticlesresultedinatotalof 17articlesregardingbasidiomycetecryopreservation.Eleven articles on cryopreservation of basidiomycetes were not includedinthisstudybecausetheywerestudiesshorterthan one-year cryopreservation. The informationcollected from thearticleswasorganizedaccordingtotheirtreatmentsand cryopreservationperiod.
Generaofcryopreservedbasidiomycetes
Thirty-seven genera were evaluated for 1-year to 2-year cryopreservationand114generaforlongerthan2-year cryo-preservation.Agaricus,PleurotusandGanodermawerethemost citedgenera(Figs.1and2).
Themostpreservedgeneraare alsothemostcultivated dueto their nutritional, functional and/or biotechnological interestsuchasAgaricusbisporus,A.subrufescens,P.ostreatus and G. lucidum. InChina, the greatest worldwideproducer, 60 different types of mushrooms are cultivated and the mostpopulargeneraarePleurotus,LentinulaandAuricularia.10 However, A. bisporus is the world most commonly culti-vated species,mainly inUnited StatesofAmerica,Poland, NetherlandandSpain.57,58Thegreatnumberofcommercially cultivatedmushroomsintheworldandthecontinuous dis-covery and introduction of new strains of basidiomycetes makethedevelopmentofcryopreservationessentialforthe maintenanceofthisbiological material.Despitethat, there isa mismatch betweenthe increase inmushroomtype in theworldproductionandthenumberoftechniquesof basid-iomycetepreservation.
Mushroomsareanimportantproductinglobaltradewith anestimatedproductionof3.4millionmetrictonsin2007. China is the biggest producer, consumer, and exporter of mushroomsintheworld,withamarketofUS$82.9million in2008and2009.10Themainproducedandcommercialized mushroomsinBrazilareA.bisporus,P.ostreatus,A.subrufescens andL.edodes.59A.bisporusmushroomsarethemostcultivated andconsumed worldwide10 and inBrazil.60 However,there arefewandconflictingdataaboutBrazilianmushroom con-sumptionandproduction,andthismakesdifficulttoprioritize cryopreservationstudies.
Inconclusion,themoststudiedgenusinshort-term cryo-preservationisAgaricusandinlong-termcryopreservationare AgaricusandPleurotusgenera.A.bisporusandA.subrufescens haveworldwideimportanceandarethemainspeciesstudied inAgaricusgenus.However,A.subrufescenshasbeenutilized incryopreservationinmostrecentstudiesduetoitsrecent commercialization,importanceandsensitivityto cryopreser-vationprocess.P. ostreatusisthemostcitedspeciesinthis genusbecauseofitsworkabilityandworldwideimportance. Cryopreservationtemperatureforbasidiomycetes
Cryopreservationat−20◦Chashadreducedsuccessin
basid-iomyceterecovery(Tables1and2).For2-yearcryopreservation at−20◦Ctherewere134treatmentsandtherewasrecoveryof
mycelialviabilityonlyin7.5%(Table1).Forcryopreservation
longerthan2yearsat−20◦Ctherewere132treatmentswith
only1.5%ofrecovery(Table2).Thus,despitebeingpossiblefor somespeciessuchP.ostreatuspreserveduntilthreeyears,52 cryopreservationata−20◦Cisnotaviabletechniqueforthe
preservationofbasidiomycetes.42,51
Freezingtemperaturesfrom−15to−60◦Careachallenge
to cellcryopreservation astheyoftenresultincryoinjuries caused byice formation, water migrationand/or ion con-centration. One of the main cell injuries to overcome in cryopreservationat−20◦Cisslow freezingthat formslong
icecrystalsinthecellintersticecausingcellularmembrane injuries.61 Besidesthe lossofcellularstructure,slow freez-ing ofexternalwatercauses cell dehydration and increase incytoplasmicionconcentration. Thisprocess reducesthe freezingtemperatureofcytoplasmturningitinahighly vis-cousconcentrationmixturewithlowmetabolicactivity but withirreversibledamagestocelland/orcellproteins.62 How-ever,Mantovanietal.52 demonstratedthatcryopreservation at−20◦CisefficientforthemaintenanceofP.ostreatusuntil
threeyearswith87%ofmycelialviabilitywhenwheatgrains areusedassubstrateand10%sucroseor4%glucoseare uti-lized ascryoprotectant.Thus, thetemperatureof−20◦Cto
preservebasidiomycetesispossibleifthecryoprotection con-ditionstoreducetheeffectofcryoinjuriesarefound.These conditionsstillneedtobedeterminedforalotofspeciesand wouldenablelong-termmaintenanceoftheviabilityof basid-iomyceteswithlowcostequipmentinthelongrun.
Themoststudiedfreezingtemperatureincryopreservation ofbasidiomycetesinseveralarticles rangedfrom −70◦Cto
−86◦C(Tables1and2).Thenumberofstudiesisgreaterfor
cryopreservationat−70◦Cto−86◦Cinaperiodrangingfrom
1yearto2years(Table1),andequivalentat−70◦Cto−86◦C
and−196◦Cwhencryopreservationislongerthan2years,and
despitethe numbers oftreatments,the studies are greater
at−196◦C(Table2).Themycelialviabilityfor
cryopreserva-tionconductedfrom−70◦Cto−86◦Cissimilarfor1-yearto
2-yearcryopreservationbutgreaterat−196◦Cwhen
cryopres-ervationislongerthan2years(Tables1and2).Thissuggests that,becauseitisatechnique(liquidnitrogen)thathasbeen establishedlonger,ithasbeenpreferablyutilizedin cryopres-ervation studies, and because ultrafreezers from −70◦C to
−86◦Careatechniquethatmorerecentlyhasbecomemore
viableeconomically, ithasbeen lessutilizedwithout being necessarilylesseffectiveinthelongrun.
Ito63andItoandYokoyama64preservednon-spore form-ingbasidiomycetesandascomycetesinaqueoussolutionwith 10% glycerol forlonger than 5years in a mechanic ultra-freezerat−80◦C.Thisprocedurecausessmallerdamagesto
thecellduetoasmallerdehydrationandmembranerupture.62 Cryopreservation at−80◦C isappropriateforseveral fungi, includingbasidiomycetes.43,65 However,tocompletely stabi-lize frozencultures,the temperature has tobe sufficiently reduced to decrease the metabolism and prevent the for-mation oficecrystals.66 According toHomolka,40 the limit temperaturetopreventicecrystalformationis−139◦Cand thisiswhyalotofculturecollectionskeeptheirsamplesat −150◦Cinultrafreezersequippedwithliquidnitrogen.
Freezing in liquid nitrogen (−196◦C) is considered one
of the best preservation methods of microorganisms in general.67 Despitethat,Colauto etal.46verifiedthatA.
sub-0 1 2 3 4 5 6 7 8 9 10 Agaricus Pleurotus Ganoderma Trametes Polyporus Pholiota Lentinula Grifola Group 1
Number of cited genera cryopreserved for 1 to 2 years
Fig.1–Mostcitedgeneraofbasidiomycetescryopreservedfor1–2years.Group1=Boletus,Coprinopsis,Cryptoporus,
Cystoderma,Entoloma,Fistulina,Flammulina,Fomes,Hebeloma,Inonotus,Lampteromyces,Langermannia,Lepista,Lycoperdon,
Micromphale,Microporus,Neolentinus,Paxillus,Pleurocybella,Pluteus,Psathyrella,Pycnoporus,Sarcodontia,Schizophyllum,
Scleroderma,Serpula,Sparassis,TephrocybeandTrichaptumgenus.
Source:24,27,41,42,45–52. Grupo 1 Grupo 2 Grupo 3 Trametes Polyporus Pholiota Ganoderma Fistulina Entoloma Pleurotus Agaricus 0 1 2
Number of cited genera cryopreserved for longer than 2 years
3 4 5 6 7 8 9 10
Fig.2–Mostcitedgeneraofbasidiomycetescryopreservedforlongerthan2years.Group1=Anthurus,Auricularia,Bolbitius,
Boletus,Bondarzewia,Bovista,Calocybe,Campanella,Ceriporia,Clavariadelphus,Coprinopsis,Coprinus,Cryptoporus,Cystoderma,
Hebeloma,Hymenochaete,Irpex,Laccaria,Lacrymaria,Langermannia,Leucocoprinus,Leucopaxillus,Lycoperdon,Macrolepiota,
Microporus,Neolentinus,Pachykytospora,Strobilurus,Stropharia,TephrocybeandVolvariellagenus.Group2=Aurantioporus,
Bjerkandera,Calvatia,Clitopilus,Coniophora,Coriolopsis,Creolophus,Cyathus,Daedalea,Daedaleopsis,Dichomitus,Faerberia,
Fomes,Gloeophyllum,Gymnopilus,Hapalopilus,Hericium,Hohenbuehelia,Hypholoma,Hypsizygus,Ischnoderma,Kuehneromyces,
Laetiporus,Lampteromyces,Laricifomes,Lentinellus,Lentinus,Lenzites,Lyophyllum,Meripilus,Merulius,Micromphale,Onnia,
Panellus,Phallus,Phanerochaete,Phlebiopsis,Piptoporus,Psilocybe,Pyrofomes,Rhodocybe,Rhodotus,Rigidoporus,Sarcodontia,
Scleroderma,Serpula,Sparassis,Stereum,Tubaria,Tyromyces,Vascellum,XerulaandXylobolusgenus.Group3=Abortiporus,
Agrocybe,Antrodia,Armillaria,Clitocybe,Collybia,Flammulina,Fomitopsis,Grifola,Inonotus,Lentinula,Lepista,Oudemansiella,
Paxillus,Phaeolus,Phellinus,Pleurocybella,Pluteus,Psathyrella,Pycnoporus,SchizophyllumandTrichaptumgenus.
Source:27,41–43,51–56.
rufescens strains respond differently to cryopreservation at −196◦C,presentingarecoveryofmycelialviabilityoftwoout
offivestrains.Forthoseauthors,thefastfreezingofA. sub-rufescensmyceliainliquidnitrogenisnotthemostappropriate methodtopreservethis species.Eichlerová et al.68 verified nosignificantlossoflaccaseactivityinthesamplesof Tram-etesversicoloraftershort-time(24h)storageat−80◦Cbutthe
storage in liquid nitrogencaused 10.6–21.7% laccase activ-itydecrease.Theauthorsdescribedthatcontrolledfreezing inliquidnitrogen,usingacoolingrateof1or3◦Cpermin, reduced laccase activity changes from 1.47 to 18.42%after short-time storage depending on the kind and concentra-tion ofthecryoprotectant. After6monthsstorageinliquid nitrogen,adecreaseoflaccaseactivityof21–44%wasfound;
Table1–Freezingtemperature,treatmentswithmycelial viabilityrecovery≥75%,andnumberoftreatmentsof basidiomycetessubmittedtodifferentcryopreservation temperaturesfrom1to2years.
Freezing temperature Mycelialviability treatments(%) Numberof treatments Source −20◦C 7.5 134 42,47–52 −70◦Cto86◦C 87.5 266 24,27,41,42,45, 47–49,51,52 −196◦C 92.9 98 24,46
Table2–Freezingtemperature,treatmentswithmycelial viabilityrecovery≥75%,andnumberoftreatmentsof basidiomycetessubmittedtodifferentcryopreservation temperaturesforlongerthan2years.
Freezing temperature Mycelialviability treatments(%) Numberof treatments Source −20◦C 1.5 132 42,51,52 −70◦Cto−86◦C 62.6 214 27,41,42,51,52 −196◦C 85 698 43,53–56
however,whenpreservedat−80◦C,laccaseactivitydecrease
was from 19 to28%. Thus,fast freezing in liquid nitrogen cancausecryoinjuriesandtheutilization ofthis technique mustberigorouslyfollowed.Coolingrateisaparameterthat strongly affects the viability ofliving cells during freezing process.68,69 Thedamageprincipleis thatcrystallizationof waterinextracellularmediumincreasesinstantaneously con-centrationofinternalcytoplasmandthe osmoticpressure, damagingtheenzymaticactivity.69Anotherinterestingaspect isthattherecoveryofviabilityafterfreezingat−80◦Cisfaster
thaninliquidnitrogenwhichmaytakedaysorweeks.70 Cryopreservationinliquidnitrogenisstillaneffectiveway topreserveseveralorganisms,includingbasidiomycetesthat cannotbelyophilized,atleastnotyet.Themaindisadvantages ofliquidnitrogenarethe highcostofequipment,constant refilling ofliquid nitrogen(every two days), separate large space forstorage,and the needof constantsurveillance.44 However,duetotheriskofbiologicalmateriallossbyrupture ofcontainers,othertechniquesmustbeutilizedto guaran-teethemaintenanceofculturecollections.Cryopreservation at−80◦Cinmechanicultrafreezersisalowercostalternative
andthatcanbecoupledtoanautomatedenergygeneratorin caseoffailureofelectricenergysupply.Asecondbackup ultra-freezershouldbeconsideredincaseofmechanicfailureofthe equipment.Therefore,itispossibletoobtainsimilarresults oflong-termcryopreservationforbasidiomycetes,mainlyin centerswherethesupplyofliquidnitrogenisnotregular.
Fewstudiesreportbasidiomycetecryopreservationby vitri-ficationorencapsulation.71,72Inthevitrificationsystem,the vitrification solutionsurrounds the cells forming an amor-phousglassandthispreventscryoinjuries.Samplesmaybe rapidlycooled in liquidnitrogen without asystem to con-trolcoolingrate.Intheencapsulationprocess,thecellsare embeddedincalciumalginatebeadspriortocryopreservation. Theutilization ofencapsulation has twomain benefits: firstly,thewatercontentofcellscanbereduceddecreasing theriskoficedamages orconcentrationeffectsduringthe coolingstage of the procedure;secondly, it allowscells to
beeasily handledand manipulatedbyproviding asuitable suspending matrix.73 Wood et al.71 reported simultaneous preservationoforchid seedsandits fungalsymbiont, Cera-tobasidiumcornigerum,usingalginate encapsulationwithno adverseeffectsaftercryopreservation.Serpulalacrymanswas immobilizedoncalciumalginatebeadsandcryopreservedat −20◦Cforonemonthwithsuccesscomparedtocontrol.72
In conclusion, cryopreservationat−20◦C isstill a
chal-lenge, mainly in long term. However,P. ostreatus has been cryopreserved successfully for 3 years when wheat grains were used assubstrate and sucroseor glucoseas cryopro-tectant.Inshortterm,cryopreservationbetween−70◦Cand −86◦C and −196◦C has been equivalently successful on
mycelialviabilityrecovery,butinlongterm,cryopreservation at −196◦C hasbeen 30% greater than between −70◦C and
−86◦C,althoughthenumberoftreatmentsat−196◦Cisthree
timesgreater.Fewstudieshaveevaluatedbasidiomycete cryo-preservationusingvitrificationandencapsulationtechniques, openingnewpossibilitiesforthepreservationofthisbiological material.
Substratesforbasidiomycetecryopreservation
Thesubstratesutilizedformycelialgrowthbefore cryopreser-vationareseveralsuchasagar-basedculturemedia,mainly potatodextroseagar(PDA)andmaltextractagar(MEA),upto cerealgrainsorperlite-based(Tables3and4).Themostcited substratetreatmentsforbasidiomycetescryopreservedfrom 1to2yearswerethosewithgrains,perlite,agar,sawdust,and grains andperlite were themostrecentlytestedin greater amounts (Table 3).Themostcitedsubstratetreatmentsfor basidiomycetes cryopreservedforlonger than 2years were thosebasedonagar,wort,grainandsawdust(Table4). Proba-bly,theagar-basedsubstrateispredominantbecausetheyare establishedproceduresandarealwaysusedascontrolinstead oftreatmentvariations;however,newtreatmentswithgrains havecomeupasanalternative(Tables3and4).
Wheatgrainhasbeeneffectiveasasubstrateeven with-out cryoprotectant to maintain the mycelial viability after cryopreservation(Tables3and4).Itispossiblethatthe pos-itive effect ofwheat grain is related tocarbohydrates and proteins that are capableof bonding water to the grain,74 reducingfreewaterandformationofintracellularicecrystals. Anotherpossibilityisthatthewheatgrainstructure, consist-ing ofacapillaritynetwork,providesphysicalprotectionto themycelium.51MataandRodríguez-Estrada75evaluatedthe viabilityofA.bisporusinwheatgrainswithandwithout glyc-erol asacryoprotectantand reportedmycelialgrowtheven intheabsenceofthecryoprotectant,suggestingthatwheat grainshavephysicalattributesthatallowmyceliatosurvive liquid nitrogen cryopreservation process. Another possibil-ity isthataspecificcompound intheseed, suchasstarch or sugar, might act as a non-permeating cryoprotectant,76 thereby reducing the injuriesassociated withfreezing and thawing.
Mantovanietal.52reportedthemaintenanceofP. ostrea-tusviabilitygrowninwheatgrainsandcryopreservedfor3 yearsat−20◦Cand−70◦C.Anothergenussuchas
Rhizocto-nia hasbeen preservedformorethan 10years inseedsof oats,barley,wheat,rye,milletandsorghum.77Mataetal.56
Table3–Substrate,treatmentswithmycelialviabilityrecovery≥75%,andnumberoftreatmentsofbasidiomycetes submittedtodifferentsubstratesfor1–2yearcryopreservation.
Substrate Mycelialviabilitytreatments(%) Numberoftreatments Source
Agar
Groundwheatgrain 0 10 50
Potato-dextrose 26.9 26 42,47,52
Malt-extract 42.4 33 41,46,50,51
Groundhardendospermwheatgrain+glycerol 0 6 51
Groundhardendospermwheatgrain+glycerol 16.7 6 51
Wheatgraingroundsemi-hardendosperm+glycerol 33.3 6 51
Groundsemi-hardendospermwheatgrain+glycerol 16.7 6 51
Groundsemi-hardendospermwheatgrain+glycerol 33.3 6 51
Groundhardendospermryegrain+glycerol 33.3 6 51
Groundhardendospermryegrain+glycerol 33.3 6 51
Barleyflouryeastextract+sucrose 37.5 8 49
Grain
Ricewithhull 41.7 12 45,47
Ricewithouthull 54.2 24 42,52
Oat 50 24 42,52
Wheat 75 24 42,52
Hardendospermwheat 33.3 6 51
Semi-hardendospermwheat 33.3 6 51
Hardendospermwheat+glycerol 33.3 6 51
Semi-hardendospermwheat+glycerol 33.3 6 51
Sorghum 50 12 48
Hardendospermrye 33.3 6 51
Hardendospermrye+glycerol 33.3 6 51
Millet 54.2 24 42,52
Perlite
Wort+5%glycerol 96.8 155 24
Sawdust
Ricebran+10%glycerol 100 69 27
reported 100% mycelialviability of Pleurotus spp. grown in sorghumandfrozenwith10%glycerolforeightyearsin liq-uidnitrogen.Tanakaet al.51 showedthat thewholegrains ofhard endospermwheathadthe highestmycelialgrowth recovery(87%)aftertwo-yearcryopreservationat−70◦Cthan onagargroundhardendospermwheat.Theydemonstrated that grain capillary physical structure is more important than grain chemical composition in substrate for keeping mycelialviabilityaftercryopreservation.Thegraincapillary net provides reduced physical space that limits the pres-enceofwatermolecules78,79 and protectsmyceliafrom ice formation. Although the capillarity in the substrate is a fundamentalfactorincryopreservation,thereisstillthe pres-ence of anti-freeze proteins produced by hard endosperm wheatgrains andusedasanalternative forA.subrufescens cryopreservationat−70◦C.51Grainshavebeenworkinglike a preservation substrate in cryopreservation even without cryoprotectants.
Perlitewithwortand5%glycerol,andsawdustwithrice bran and glycerol, also presented treatments with greater mycelial viability from 1 to 2 years and after 2 years
(Tables3and4).However,thetreatmentofagar-based
cryo-preservationafter2years withwortor malt extract+yeast extract+glycerolwasverypromising(Table4).Ontheother hand,aseriesofcombinationsofagar-basedsubstratewith maltextract,wheatgrainorgroundyeastwerenoteffective
fortherecoveryofmycelialviabilityafter2yearsof cryopres-ervation(Tables3and4).
The cryopreservationmethod using perlite54 withmore than 400 tested strains, or sawdust27 with more than 30 evaluated strains or sorghumgrain56 withfour strains, for morethan 5years,was effectivetomaintain mycelial via-bilityofbasidiomycetes.Perliteistheonlyvolcanicmineral ofaluminumsilicatethatretainslargequantitiesofwaterto release asneeded.Theprotocolwithperlitecanbeusedto cryopreservegroupsoftaxonomicallydifferentfungi,besides basidiomycetes,includingyeast,26andisasanalternativefor otherfungithatdidnotsurviveroutineproceduresof preser-vation.
Inconclusion,substratessuchasperlite+wort+glycerol, followed by sawdust+rice bran+glyceroland cereal grains (wheat,rice,sorghum,oatormillet),withoutcryoprotectant added tothe substrate,havebeen more effectivein short-termcryopreservation,mainlywhencomparedtoagar-based culturemedium.Long-termcryopreservationhavebeenmore effective in substrates such as perlite+wort+glycerol, fol-lowedbysawdust+ricebran+glycerol,sorghumgrainswith glycerol, and wheat grains with or without cryoprotectant addedtothesubstrate.However,theagar-basedsubstrateof malt-extract+yeast-extract+glycerolhasbeenalsoeffective inlong-termcryopreservationwhenyeastandglycerolwere addedtothesubstrate.
Table4–Substrate,treatmentswithmycelialviabilityrecovery≥75%,andnumberoftreatmentsofbasidiomycetes submittedtodifferentsubstratesforlongerthan2yearcryopreservation.
Substrate Mycelialviabilitytreatments(%) Numberoftreatments Source
Agar
Potato-dextrose 22.2 36 42,52
Malt-extract 27.8 18 41,51
Malt-extract+glycerol 0 6 51
Malt-extract+yeastextract+glycerol 93.2 176 53
Groundhardendospermwheatgrain 0 6 51
Groundhardendospermwheatgrain+glycerol 0 6 51
Groundsemi-hardendospermwheatgrain 0 6 51
Groundsemi-hardendospermwheatgrain+glycerol 0 6 51
Groundhardendospermryegrain 0 6 51
Groundhardendospermryegrain+glycerol 0 6 51
Wort 61.3 243 53,54
Grain
Ricewithhull 27.8 36 42,52
Oat 27.8 36 42,52
Wheat 60.9 46 42,43,52
Hardendospermwheat 16.7 6 51
Semi-hardendospermwheat 16.7 6 51
Hardendospermwheat+glycerol 16.7 6 51
Semi-hardendospermwheat+glycerol 16.7 6 51
Hardendospermbarley 0 6 51
Hardendospermbarley+glycerol 0 6 51
Millet 36.1 36 42,52
Sorghum 100 4 56
Sawdust
Ricebran+10%glycerol 97.1 70 27
Perlite
Wort+5%glycerol 100 265 54,55
Cryoprotectantsforbasidiomycetecryopreservation
A cryoprotectant is utilized to reduce cryoinjuries in the cellduringfreezingand cryopreservation.Themechanisms inwhich these cryoprotectants act havebeen reviewedby Meryman,80 Calcott,81Heckly82andHubálek.32Itisbelieved that the cellular membrane isthe primarylocation ofthe injuryduetoformationoficecrystalsduringfreezing, caus-ing physicalstress and cellularrupture.83 Furthermore,ice concentratessolutes that cause chemical stress in cellular molecules.32
Differentcryoprotectantsareutilizedincryopreservation ofbasidiomycetes,and themostcitedtreatmentsuse glyc-erol,followedbydimethylsulfoxide,glucoseandsucrosefor cryopreservation after 2 years (Tables 5 and 6). Hubálek32 classifiedthecryoprotectantsaccordingtotherateof pene-tration:thosethatpenetrate quickly,usuallywithin30min, theonesthatpenetratemoreslowly,andnon-penetratingor non-permeatingcompoundsthatcauseextracellular cryopro-tectionwhenpresentatconcentrationsof10–40%.Also,the authorclassifiedcryoprotectantsasthosethatpenetrateonly thecellwallandnotthe cytoplasmicmembraneandthose thatpenetratebothcellwallandcytoplasmicmembrane.
Glycerol isthe most citedcryoprotectant intreatments withcryopreservationof1–2yearsanditwasalsothemost effectivetokeepmycelialviabilityaftercryopreservation, dif-ferentlyfrom dimethylsulfoxide thatisnot soeffective to basidiomycetes when compared with glycerol (Table 5). In general,thosewithoutcryoprotectantsthathadcerealgrains
Table5–Cryoprotectant,treatmentswithmycelial viabilityrecovery≥75%,andnumberoftreatmentsof basidiomycetessubmittedtodifferentcryoprotectants from1-2yearcryopreservation.
Cryoprotectant Mycelialviability
treatment(%) Numberof treatments Source Ultrapurewater 0 32 50,51 Dimethylsulfoxide 1% 59 22 41,42,52 10% 40 5 46 Maltextract 4% 50 10 42 5% 58.3 12 41,52 Glycerol 5% 83.6 201 24,47,50–52 6% 100 2 41 10% 47.1 34 42,45,48,49 Glucose 4% 50 26 41,42,50,52 Polyethyleneglycol 6% 35 20 42,52 8% 100 2 41 Sucrose 10% 65 20 42,52 15% 33.3 6 41,50 Withoutcryoprotectant 34.2 38 48,50,51
Table6–Cryoprotectant,treatmentswithmycelial viabilityrecovery≥75%,andnumberoftreatmentsof basidiomycetessubmittedtodifferentcryoprotectants forlongerthan2yearcryopreservation.
Cryoprotectant Mycelialviability
treatments(%) Numberof treatments Source Ultrapurewater 0 28 51 Dimethylsulfoxide 1% 28.1 32 41,42,52 Maltextract 4% 20 20 42 5% 33.3 12 41,52 Glycerol 5% 91.2 479 51–55 6% 0 2 41 10% 68 337 27,42,53,54,56 15% 100 10 43 Glucose 4% 46.9 32 41,42,52 Polyethyleneglycol 6% 10 30 42,52 8% 0 2 41 Sucrose 10% 50 30 42,52 15% 100 2 41 Withoutcryoprotectant 0 28 51
as substrate (preservation substrate) or when the number oftreatments was not representative, even with a greater mycelialviability,werenotconsideredinouranalysis.Tanaka etal.51suggestedthattheuseofglycerolinassociationwith wholegrainscouldimprovethemycelialviabilityofA. sub-rufescenscryopreservedat−80◦Cand−20◦C.
Themostcitedcryoprotectantintreatmentswith cryopres-ervationafter2years,glycerol,wasalsothe mosteffective for the maintenance ofmycelial viability after cryopreser-vation;however,other cryoprotectantssuchassucroseand glucosearegoodalternativesthathaveshowntobeefficient whiledimethylsulfoxidehasshownlowerefficiency(Table6). Glycerolisclassifiedasapenetratingorintracellular cryopro-tectantandhasthepropertytobonditselftowatermolecules, minimizingtheformationandsizeoficecrystalsaswellas reducingsoluteconcentrationsintheextracellularor intracel-lularmedium.32,84Thiscryoprotectantpenetratesthecellular membranethroughpassivediffusion,gettingintothe mem-braneaswellasinthecytoplasm85;itsprotectiveeffectsare representedby colligativeproperties, reducing the freezing pointwithconsequentreductionofelectrolyteconcentration inthenon-frozenfractionofthesample.86
Althoughglycerolisthemostutilizedcryoprotectantinthe preservationoffrozenbasidiomycetes, it can haveadverse effectslikephysicalandchemicalalterations,ruptureof plas-maticmembrane,removalofimportantmembraneproteins, andit caninducemodificationsinthe stabilityofthelipid structure.87,88
Theconcentrationofglycerolrangedfrom5to15%,with greater mycelialviability at5%forcryopreservationafter2 years(Table6).ItoandNakagiri89examinedtheviabilityof939 strainsofbasidiomycetescryopreservedat−80◦Cforone,five
and15yearswitheffectivemycelialviabilityof88%ofthese strainsinagarplugswithmyceliasubmersedin cryoprotec-tantsolutionof10%glycerol.Tanakaetal.51suggestedthatthe useofglycerolinassociationwithwholegrainscouldimprove themycelialviabilityofA.subrufescenscryopreservedat−80◦C
and −20◦C.Colautoet al.41reportedthatconcentrationsof 10–30%glycerolcausedinhibitionofmycelialgrowthofA. sub-rufescens.HoweverMataetal.56reportedthat10%glycerolwith sorghumgrownwithPleurotusspp.hadmycelialviabilityof aftereightyearsofcryopreservationinliquidnitrogen.
The cryoprotective effect of glycerol is probably due to the capacity of this cryoprotectant to penetrate the cellular wall and the plasmatic membrane and to link waterintheinterstice betweenthe wallandtheplasmatic membrane,90 increasing the membrane elasticity, which allows the molecular accommodation during the volume expansioninfreezing.60,91
Sucroseaswellasglucoseshowedeffectivemycelial viabil-ityincryopreservedtreatmentsofmorethan2years(Table6). Colautoetal.45evaluatedA.subrufescenscryopreservationat −70◦Cusingdifferentcryoprotectants.Forthoseauthors,the
cryoprotectiveagent–andnotthefreezingprotocol–wasthe mostimportantvariableforeffectivemycelialviabilityafter cryopreservation.Long-termcryopreservation(4years)ofA. subrufescenswaseffectivewhensucroseorglucosewasusedas cryoprotectant,regardlessofthefreezingprotocol,and glyc-erol,polyethyleneglycol,andmalt extractwereineffective, dimethylsulfoxidewaseffectiveonlywhenaslowfreezing protocolwasused.41However,forcryopreservationof1year, all cryoprotectantswere effective tokeepmycelialviability ofA.subrufescens.45Sucroseandglucosearenotableto dif-fusethemselvesthroughtheplasmaticmembrane,butthey increasetheexternalosmoticstrengthandpromotethe cellu-lardehydration,reducingtheformationoficecrystalswithin thecell.32Thesesugarscanbeutilizedasasourceofcarbonin therecoveryofthecellulargrowth.Sucroseisfrequentlyused incryopreservationofmicroorganismsatconcentrationsfrom 1to68%(averageof10%).32Colautoetal.41reported concen-trationsof5,15 and30%sucroseincultivationmediawith inhibitionofmycelialgrowthafter15%.
Cryoprotectant agents can be utilized as an additive to the substrate. Tanaka et al.51 added glycerol to the sub-stratebeforecryopreservationofA.subrufescenswitheffective increaseofmycelialviabilityaftertwo-yearcryopreservation at−70◦C.Theutilizationofcryoprotectantsasasolutioninthe
cryotubeoranadditiontothesubstrateisimportantto long-term basidiomycete cryopreservation. However, the choice of concentration and cryoprotectant depends on the cell type, strain, mycelialage, substrate,cultivation conditions, amongotherfactorsthataffectthepermeabilityandelasticity ofmembranes.41 Despitetheimportanceofthe cryoprotec-tantforthemaintenanceofcellularviabilitythroughoutthe cryopreservation,thiscompoundcancausemycelialgrowth inhibition.Therefore,thetypeofcryoprotectant,the concen-trationandinhibitionoradverseeffectsonmycelialgrowth must be defined for each case, depending on strain and thecryopreservationmethod.Colautoetal.41 evaluatedthe adverseeffectsofcryoprotectantsusedincryopreservationof A.subrufescensandreportedthattheincreaseofthe concen-trationinhibitmycelialgrowth.Accordingtotheseauthors,
dimethyl sulfoxide and glycerol inhibited mycelial growth, mainlydimethylsulfoxideatconcentrationsof2.5%and5%, completelyinhibitingmycelialgrowth,butsucrose,glucose, polyethyleneglycolandmaltextractshowedlowinhibitionof mycelialgrowth.
In conclusion, the use ofcryoprotectants inshort-term cryopreservation has been effective mainly with glycerol, followed by sucrose, dimethyl sulfoxide, malt extract and glucose. However, in long-term cryopreservation the most effectivecryoprotectanthasbeenglycerol,followedbysucrose andglucose.Dimethylsulfoxidehasnotbeeneffectivein long-termcryopreservationforbasidiomycetes.
Cryopreservationperiodforbasidiomycetes
Theintervalof1to<2yearsofcryopreservationhadthe great-estnumberofarticlesandtheinterval of5yearsorlonger hadthesmallestnumberofarticles(Table7).Thisisdueto thedifficultytocarryoutlong-termtreatmentswithalotof replications,runningtheriskoflosingthegeneticmaterial becauseoffailure inthe equipment andthe supply of liq-uidnitrogenduringthecryopreservation.Moreover,long-term studies demand planning and professional stability in the workplace.Therefore,agreateramountofstudiesinashorter cryopreservation timeinterval, mainly a year atthe most, iscommon.25,28,45,46,48,55,92Somecryopreservationbetween2 andlessthan5yearswereeffectiveforseveralbasidiomycetes whereas others were effective for 5 years or longer as 15 years(Table7).However,eachspeciesand eveneachstrain ofbiotechnological importancemusthaveaprotocoland a specificcryopreservationmethodbecauseitisnotpossibleto meetallthe specificationsforallbasidiomyceteswithonly onemethod.39,93 Thecryopreservationofmorethan5years withbasidiomycetesisachallenge.Besidesthe cryopreser-vationperiodvaryingaccordingtoeachmethodandspecies, themethodhastoprovidethelong-termmaintenanceofthe mycelialviabilityandthegeneticandphysiologicalstability ofthefungus,andthesubstrateforbasidiomyceterecovery afterfreezing.
Thenumberofbasidiomycetecryopreservationstudiesis greaterforperiodsshorterthan5years(Table7).Onlythree articles successfullydescribed basidiomycete cryopreserva-tion for 5 years or more, probably due to the difficulty of maintainingaculturecollection forso longor evendueto techniquefailuresafter5years.Thescarcityof cryopreserva-tionreportsafter5yearsshowsthedifficultyofmaintaining studiesforsuchlongperiodsandthelimitsofthetechnique.
Table7–Cryopreservationperiod,treatmentswith mycelialviabilityrecovery≥75%,andnumberof treatmentsofbasidiomycetessubmittedtodifferent cryopreservationperiods. Cryopreservation period Mycelialviability treatments(%) Numberof treatments Source 1to<2years 72.6 456 23,26,40,41,44–51 2to<3years 67.3 432 26,41,50,52 3to<5years 74.2 519 26,40–42,51,53 5yearsorlonger 100 48 26,54,56
Inconclusion,basidiomycetecryopreservationformorethan 5yearsisstillachallenge.
Cultivationmediumandtemperaturefortherecoveryof cryopreservedbasidiomycetes
Thethawingtemperatureandthecultivationmediumutilized inthe recoveryofbasidiomycetesafterfreezing are impor-tantparametersinthecryopreservationprocess.Thethawing temperaturehasbeenstandardizedandutilizedsuccessfully throughtheimmersioninwaterat30◦Cfor15min.41,42,45,51,52 Thechoiceoftherecoverycultivationmediumisgenerally basedontheoneinwhichthefungusisusuallyinitiallygrown beforefreezing,butitisnotalwaysthebestchoice.
PDA cultivation medium is the most utilized in frozen basidiomyceterecovery.27,47,48,52Thesecondmostutilized cul-tivationmediumisMEA,41,43,45,56followedbyotheragar-based culturemediasuchaswort-agar,24,53MEYA(maltextract,yeast extract, glycerol and agar)53 and MGL1 (barley flour, yeast extract,sucroseandagar).49Tanakaetal.(unpublisheddata) verifieddifferencesinthemycelialviabilityofA.subrufescens after5yearsofcryopreservationat−70◦CinMEAwith
differ-entconcentrationsofagarandverifiedthatthewateractivity inthecultivationmediumaffectstherecoveryofmycelial via-bility.
Inconclusion,culturemedia,temperatureandother inher-entaspectstotherecoveryofcryopreservedbasidiomycetes arestilllittlestudiedwithagreaterconcentrationofstudies intheinitialandintermediatestagesofthecryopreservation process.
Geneticandphysiologicalstabilityofbasidiomycetesafter cryopreservation
The expansion of the biotechnological applications of basidiomycetes requires maintenance of the physiologi-cal, morphologicaland therapeuticcharacteristics, and the genomic stability after thawing of the biological material, besides the long-term preservation of the mycelial viabil-ity.Allarticlesinthisreviewevaluatedthemycelialviability of basidiomycetes after thawing, 23.5% ofthem also eval-uated characteristics like the production of enzymes such aslaccaseandperoxidasemanganese,24,53–55 29.4%ofthem characteristics like mushroom production,27,43,47,48,56 11.7% of them characteristics such as the genetic stability with theutilization ofmolecularmarkers43,55 and 17.6%ofthem macro andmicromorphologicalcharacteristics suchas tex-ture,coloringanddensityofmycelium,presenceorabsence of connection clamps among others.24,54,55 Only one arti-cle analyzed biomass production, exopolysaccharides and intrapolysaccharides.49
Overall,thearticleshaveshownthatmycelialviabilityand morphological,geneticorphysiologicalcharacteristicsmaybe preservedbyseveralcryopreservation methodswhilesome others are lesseffective. Eichlerováand Homolka24 didnot observe loss in the laccase production for most analyzed basidiomycetes. Homolka et al.53 reported similar results whenevaluating250strainsofbasidiomycetesinwhichonly twodidnotkeeptheactivityoflaccaseafterthawing.Positive
resultsregardingthemaintenanceoftheenzymeproduction activitywerealsoobservedbyHomolkaetal.54
Singhetal.43andHomolkaetal.55assessedthemycelial viabilityandotherparameterslikemaintenanceof morpho-logicalandenzymaticcharacteristicsandgeneticstabilityof cryopreservedbasidiomycetesanddidnotobservechangesin thesecharacteristics.Homolkaetal.55 verifiedthe morpho-logical,physiologicalandgeneticstabilityof30cryopreserved basidiomyceteswithoutfindingsignificantdifferences.Zaghi Junioret al.(unpublished data)didnotverify alteration by RAPDmolecularmarkersinthepatternofampliconsamong A.subrufescensstrainscryopreservedfortwoyearsat−75◦C, either.
Inconclusion,mycelialviabilityanalysisofcryopreserved basidiomyceteisstillthemainandmostimportantwayto ver-ifytheefficacyofthetechnique.However,otheraspectssuch asanalysisofrecoveryperiodafterthawing,morphological, physiologicalandgeneticstabilityareimportantparameters ofanalysisofthebiologicalmaterialaftercryopreservation. For genetic stability analysis, techniques that evaluatethe greaterpartofthegenomearemorerepresentativeasRAPD indetrimentofITS.However,duetothelongtimeofthestudy and the number of treatments and the microbial isolates, theevaluationofall characteristicsbecomesimpracticable. Therefore,reducingtheamountofanalyzesforoneofeach typesuchasviability,recoveryperiod,morphological, physio-logicalandgeneticstabilityareapossibleanalysisuntiloneof thetreatmentsoncryopreservationbecomesmoreeffective, and then reducing the number oftreatments and increas-ingthenumberofanalysis.Itisrecommendedtoreducethe numberofspeciesperstudyasthereisnouniversal cryopres-ervationtechniqueforbasidiomycetes.
Conclusion
Inthelast20yearsofbasidiomycetecryopreservation stud-ies,only37generahavebeenpublishedandthemoststudied genera in short-term cryopreservation are Agaricus, Pleuro-tusandGanoderma,and114generahavebeenpublishedand themost studiedgenerainlong-term cryopreservationare Agaricusand Pleurotus. Cryopreservationat −20◦C is still a
challengebutwheatgrainscouldbeanalternativetoimprove long-termcryopreservation.Cryopreservationbetween−70◦C and−86◦Cand−196◦Careequivalentlyinshort-and
long-termcryopreservationbutinlongtermat−196◦Cthereisa
greater numberofpositiveresults. Themosteffective sub-strates are perlite-based, sawdust-based and cereal grains in short- and long-term cryopreservation; however, agar-basedsubstrate ofmalt andextractwithglycerolhasbeen reported as effective in long-term cryopreservation. Glyc-erol mainly, but sucrose, dimethyl sulfoxide, malt extract andglucosehasbeeneffectivecryoprotectantsinshort-term cryopreservation.However,inlong-termcryopreservationthe mosteffective cryoprotectant has been glycerolfirstly and thensucroseandglucose.Dimethylsulfoxidehasnotbeen effectivein long-termcryopreservation forbasidiomycetes. Basidiomycetecryopreservationformorethan5yearsisstill achallenge.Culturemedia,temperatureandotherinherent aspectstotherecoveryofcryopreservedbasidiomycetesare
stilllittlestudiedwithagreaterconcentrationofstudiesin the initialand intermediatestages ofthe cryopreservation process. Mycelial viability analysis of cryopreserved basid-iomyceteisstillthemainandmostimportantwaytoverify the efficacy of preservation. However, other aspects such asanalysisofrecoveryperiodafterthawing,morphological, physiologicalandgeneticstabilityareimportantparameters ofanalysisofthebiologicalmaterialaftercryopreservation.It isrecommendedtoreducethenumberofspeciesperstudy asthereisnouniversalcryopreservationtechniquefor basid-iomycetes.
Conflicts
of
interest
Theauthorshavenotdeclaredanyconflictsofinterest.
Acknowledgements
Theauthors thankParanaense University,Posgraduate Pro-graminBiotechnologyAppliedtoAgricultureofParanaense University,Coordenac¸ão de Aperfeic¸oamento dePessoal de NívelSuperior(CAPES)andConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPq).
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