Subject: In vitro boletes

From: [email-masked] (Richard Winder)
Date: Wed, 24 May 1995 17:26:49 GMT

Hello,

A number of people have requested further information from me regarding
my in vitro culture of B. edulis primordia.  So, I hope I am not flogging
this to death, but I decided that the best thing to do would be to post
a synopsis here.  This is meant to be a fun, quick report and not a
comprehensive overview- there's probably lots of information out there that
I've missed.  So, if you have anything to add, please be my guest!

			********************

    In vitro culture of Boletus edulis primorida
    
    Richard S. Winder
    Pacific Forestry Centre
    506 W. Burnside Rd.
    Victoria, B.C.  V8Z 1M5  CANADA
    
    -Disclaimer
    
    Mention of trade names does not imply endorsement of a particular 
    product, nor does it imply criticism of any similar products not 
    named.  This posting may be distributed freely by anyone, as long as 
    this disclaimer remains in place.  -RSW

    -Introduction
    
    Although they are ectomycorrhizal, it is possible to grow members of 
    the boletaceae in axenic culture and produce their associated 
    fruiting bodies or primordia (Table 1).  The usual method involves 
    the use of Hagem-Modess medium (Modess, 1941) or some modification of 
    it (McLaughlin, 1970), in containers ranging in size from test tubes 
    to jars.  This report outlines an in vitro method for generating 
    primodia of Boletus edulis using potato dextrose agar (PDA), a more 
    standard mycological medium.
    
    Table 1. Some reports of in vitro production of fruiting bodies for 
    the boletaceae.
    
    ---------------------------------------------------------------
    Fungi		    Citation			Result
    ===============================================================
    Boletus spp.	    Pantidou, 1961a		Primordia
    			    McLaughlin, 1970		Mushroom
    Boletus edulis	    Karpi'nski, 1961		Mushroom
    Boletus rubinellus	    McLaughlin, 1970		Mushroom
    			    Yoon & McLaughlin, 1979	Mushroom
    Leccinum spp.	    Pantidou, 1961a		Primordia
    			    McLaughlin, 1970		Mushroom
    Phlebopus lignicola	    Pantidou, 1962		Mushroom
    Phlebopus sulphureus    Pantidou, 1961b		Mushroom
    Pulveroboletus spp.	    Pantidou, 1961a		Primordia
    Tylopilus spp.	    Pantidou, 1961a		Primorida	 
    			    McLaughlin, 1970		Mushroom
    Xerocomus spp.	    Pantidou, 1961a		Primordia
    Xerocomus badius	    Pantidou, 1964		Mushroom
    Xerocumus illudens	    Pantidou, 1964		Mushroom
    ----------------------------------------------------------------
    
    -Materials and methods
    
    	Dehydrated potato dextrose agar (No. 6013-01-4 Bacto agar, Difco 
    Laboratories, Detroit, Michigan, U.S.A.) was combined (39 g/L) with 
    tap water (pH 6.9), autoclaved (15 p.s.i., 121 C) for 20 minutes, and 
    poured into 9 cm-diam. Petri plates to solidify.  A sporophore of 
    Boletus edulis (white pore stage) growing in association with grand 
    fir (Abies grandis) was collected in Sooke, British Columba in 
    September, 1995.  After peeling back the cuticle, tissue from the 
    sporophore was excised and aseptically transferred to 10 PDA plates.  
    The plates were sealed with wax film (Parafilm, American National 
    Can, Greenwich, CT, USA) and incubated cover-side-up in a controlled 
    environment chamber at 20 degrees C for eight months.  The colony 
    diameters were recorded as well as the number and diameter of 
    primorida forming in each plate.
    
    -Results
    
    	After eight months, the medium lost about 50% water content as 
    measured by the thickness of the agar.  Five plates were discarded 
    due to contamination.  The colonies did not colonize the entire 
    plate, colony diameter (largest dimension) ranged from 37-70 mm (mean 
    49 mm).  Colony edges were highly irregular and diffuse, hyphae were 
    white.  Sporophore primorida of various sizes formed in the remaining 
    plates.  The primordia where chalky white in color, with a few drops 
    of brownish liquid present on some surfaces.  Hyphae close to the 
    primordia tended to be rhizomorphic.  The primordia were composed of 
    parenchyma-like cells which appeared to be somewhat organized into 
    layers.  Most of the larger primordia developed abortive tubes on 
    their upper surfaces.  In some cases, smaller globular primordia 
    appeared on top of ther larger primordia.  Primordia were 7-32 (mean 
    19) mm in diameter, with an average of 7.1 primordia (all types) per 
    plate.
    
    -Discussion
    
    	A variety of simple undefined media could probably be used to 
    produce primordia of the boletaceae in vitro.  The method could 
    probably be improved by using a larger container for the agar.  While 
    it is premature to speculate that this method could be used to 
    generate mature sporocarps in any quantity, it could provide an 
    easily accessbile starting point for testing various ammendments and 
    other other factors which might influence sporocarp production.
    
    -Literature
    
    Karpi'nski, J. 1961. Investigation results (stage I) pertinent to the 
        cultivation of fruiting bodies of Boletus edulis Bull., in 
        artificial culture medium under laboratory conditions [In Polish, 
        English summary]; Sylwan 105:55-59.
    McLaughlin, D. 1970. Environmental control of fruitbody developement 
        in Boletus rubninellus in axenic culture. Mycologia 62:1970.
    Modess, O. 1941. Zur Kenntnis der Mykorrhizabildner von Kiefer und 
        Fichte. Symb. Bot. Ups. 5:1-147.
    Pantidou, M. 1961(a). Cultural studies of boletaceae: Gyrodon 
        meruloides and four species of Boletinus. Can. J. B. 
        39:1149-1162.
    Pantidou, M. 1961(b). Carpophores of Phlebopus sulphureus in culture. 
        Can. J. Bot. 39:1163-1166.
    Pantidou, M. 1962. Cultural studies of boletaceae: carpophores of 
        Phlebopus lignicola in culture. Can. J. Bot. 40:1313-1319.
    Pantidou, M. 1964. Cultural studies of boletaceae: carpophores of 
        Xerocomus badius and Xerocomus illudens in culture. Can. J. Bot. 
        42:1147-1167.
    Yoon, K. and D. McLaughlin. 1979. Formation of the hilar appendix in 
        basidiospores of Boletus rubinellus. Am. J. Bot. 66:870-873.

  RICHARD WINDER                    Title: Research Scientist
  Canadian Forest Service           Phone: (604) 363-0773
  Victoria, B.C.                    Internet: [email-masked]