Chapter-4 Breeding Materials

The procedure for mating type identification of the mycelium products after single spore isolation
Steps Crossing two test monokaryotic stocks on PDA plate Cutting off the mycelial fragment at the association part with two colonies with knife Observe the existence of clamp connection in case that the test species has clamp connection Cutting off a piece of mycelium fragment at the part where the dikaryotization of hyphal cells has been confirmed Preparation of crossing table to identify either one of the mating types in bipolar or tetrapolar mushrooms
Observation of the hylae of the test mycelium stock under light microscope
Observe the test mycelium colony carefully to confirm the absence of clamp connection all over the light field
(1) Single spore isolation for preparation of monokaryotic mycelia
This method is usually be applied for preparation of monokaryotic mycelium stocks of mushroom fruit bodies, the basidiospores of which can germinate at practically available level. The mycelium products are the haploid recombinants after processing through nuclear fusion between two nuclei of mother dikaryotic cells, meiosis, and basidiospore formation, and spore discharge, successively. Therefore, the products show minor genetic diversities to each others.
An example of crossing table for tetrapolar mating type of mushroom
An experimental result Rearrangement table
Dual cultures showing the barrage reaction between two colonies in a Petri dish in tetrapolar mushroom
Slide culture Fixing with aqueous mixture Washing of ethanol and acetate = 3 : 1
(Fluorescent microscope)
Double staining with DAPI and Calcoflour white
Collect spores by Centrifugation
Stood over
Dilution of spore suspension in Petri dish
Discard the supernatant
Suction of suspension into micro pipette
Drop each spore into malti-plate
① Method for preparation of dikaryotic mycelium stocks
Regeneration of vegetative mycelium from fruit body tissue by incubating a piece of tissue (obtained by cutting with knife) on slant agar medium
Mixing with 0.7%PDA soft agar
Stepwise dilution of spore suspension
Spore print
Technique for single spore isolation using micromanipulator
Spore print
Stood night for overnig ht
(2) Preparation of monokaryotic stocks by surgical isolation of terminal hyphae from dikaryotic mycelium colony
This method is based on the mycelium monokaryotization phenomenon at the terminal zone of dikaryotic mycelium colony. The nucleus of the products is the same of either one of two nuclei in the mother dikaryotic mycelium. In other words, it is possible to obtain the same genetic composition of monokaryotic stock as to one of the ancestor monokaryotic stocks without passing through sexual reproduction. The almost all of the isolated stocks contain either one type of nucleus, due to the nuclear selection rule. The product is effectively available for the improvement of the mother strain by crossing with different line of the same species.
Summary 1
1) Single cell isolation is the most conventional method for preparing monokaryotic mycelium, and the using of double layer medium in its procedure is an easy way for separating the spore germinates. 2) It is indispensable to confirm of the state of monokaryon by either the observation of clump connection or the count of nuclei by double fluorescent staining. 3) The spore isolates is the resulting products of chromosome recombination, and the isolated monokaryon carry different composition of genetic traits each others. 4) For using the monokaryotic isolates for breeding, the mating type identification is necessary process prior to hybridization, and the getting results should be rearranged to prepare the crossing table.
Technique of single spore isolation
Fruit body Collection of spore print
Spore suspension with water
Incubation for germination and colony formation Pour 0.7% soft agar mixture on to 2% PDA
In case of the species which does not form clamp connection, we have to go to confirm the nuclear state of the test stock by fluorescent staining
Staining technique with fluorescent dyes for nucleus and septum of hyphal cells
Micromanipulator assembly
Microplate culture of single spore
Germination rates of basidiospores from various mushrooms on PDA medium
The method for confirmation of monokaryotic phase of mycelium from various procedures