The initial mycelium of L. laccata, grown in MMN medium (Marx, 1969), is macerated in a blender for 30 s and added to a 250 ml flask with fresh medium (equal vol.) and allowed to grow for 3-4 days. Resultant mycelia are recovered by centrifugation and washed once with MMC (Barrett et al., 1990). The packed volume of mycelia is usually 2-4 ml. This mycelial pellet is suspended in 15 ml enzyme mix containing 5-7mgml_1 Novozyme 234 and lmgml-1 bovine serum albumin (BSA) in MMC. This enzyme mix is filter sterilized before addition to the mycelia. The mycelia are incubated in the enzyme mix for 2-4 h at 31 °C with gentle shaking (125 rpm).
Novozyme 234 is a lysing enzyme preparation from Trichoderma harzianum sold by Novo Industries. Comparable enzyme preparations from Sigma Chemical or Cayla also work well and may be substituted.
Liberated protoplasts are separated from hyphae by filtering the slurry through cotton. A wad of cotton is tamped into the bottom of a 10 ml plastic syringe. The cotton and the syringe are autoclaved before use. The cotton forms a loose filter 1 ml deep. Wet the cotton with MMC to make sure it stays in place and pour the protoplast/hyphal slurry through, letting the clear solution drip through by gravity. Centrifuge the filtrate in a table-top centrifuge at 2500 rpm to pellet the protoplasts. Wash the protoplasts twice in MMC by successive centrifuga-tions.
Resuspend the protoplast pellet in MMC and count the protoplasts on a haematocytometer. A pellet resuspended in 1 ml usually contains 1-2 x 108 protoplasts. If there are more, dilute with MMC; if there are fewer, pellet again and resuspend to achieve 1-2 x 10"8 protoplasts mr1.
In a sterile microfuge tube, incubate 10-50 fig of pAN7-l DNA with 50 fig heparin for 10-15 min. Add lOOju.1 (1-2 x 10"7) protoplasts and 50 /xl PEG solution (Yelton et al., 1984). Mix well. Incubate on ice 30-45 min. While the protoplasts are incubating, add 1 ml of the PEG solution to a sterile 15 ml tube. When the protoplast incubation period is over, transfer the protoplasts to the 15 ml tube. This step facilitates handling because the PEG solution is difficult to pipette and it is more expedient to transfer the protoplasts into the 1 ml of PEG than to pipette the PEG into the microfuge tube. Mix well and continue the incubation for 10-15 min at 22 °C.
Wash the protoplasts by centrifugation with regeneration medium (Kitamoto et al., 1988) modified to contain only 0.4 m sucrose. The usual regeneration concentration of 0.6 m is probably too high because of the high concentration of PEG. Resuspend the protoplasts in regeneration medium containing 0.6 m sucrose and allow the protoplasts to recover in this liquid medium for 5-7 days. This allows the protoplasts to regenerate and permits early expression of the hpt gene in transformants before plating.
Plate the regenerates on MMN medium containing 2% agar and 200 /Agml-1 hygromycin B (HmB). Plates are incubated at 22 °C in the dark and observed after at least 1-2 weeks.
By this protocol between 5 and 50 putative transformants are recovered. Transfer colonies that grow on 200 fig ml-1 HmB to fresh plates with 200 fi ml-1 and then to MMN agar medium containing higher levels of the drug. Many of the isolates fail to grow at higher concentrations of the antibiotic. Isolates that grow on 500 and 1000 /igml-1 are selected for further study. Hygromycin B is also a mutagen and some isolates recovered on 1000 fig ml1 of HmB contain no plasmid sequences when examined by Southern (1980) or dot blot (Mohr, 1989) analyses of fungal DNA probed with 32P-labelled pAN7-l.
Genomic DNA is isolated from selected colonies by growing gram fresh weight quantities of mycelium in MMN medium with HmB or by growing colonies on 0.45 ¿¿m filters (Millipore, nitrocellulose) placed on top of agar medium in a Petri dish. The colonies peel off the nitrocellulose and are free from agar contamination. The mycelium must be frozen and lyophilized, and is then very sensitive to lysis in sodium dodecyl sulphate (SDS). Use 1% SDS, 0.05 m EDTA pH 8, 25 aigml"1 proteinase to lyse the cells.
Either the procedure of Zolan and Pukkila (1986) is followed or extractor columns (Molecular Biosystems, Inc.) are used to purify the fungal DNA. The DNA usually forms a flocculant rather than a spoolable precipitate in cold ethanol, but it seems to be of high molecular weight upon analysis by electrophoresis in 0.8% agarose gels (Barrett et al., 1990).
1. Media and buffers MMN medium (Marx, 1969)
0.3% malt extract
25 /xg litre "1 thiamine-HCl 1.2 ¿ig litre-1 FeCl3
MMC buffer (Barrett et al., 1990)
0.5 m mannitol
50.0 mM CaCl2
Regeneration medium (Kitamoto et al., 1988)
0.05% yeast extract
0.6 m sucrose
PEG solution (Yelton et al., 1984)
60% polyethelene glycol 4000
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