Isolation of fungal homokaryotic lines from heterokaryotic transformants by sonic disruption of mycelia
Citation
Bashi, Z.D., Khachatourians, G., Hegedus, D.D. (2010). Isolation of fungal homokaryotic lines from heterokaryotic transformants by sonic disruption of mycelia. BioTechniques, [online] 48(1), 41-46. http://dx.doi.org/10.2144/000113243
Abstract
Fungal hyphae - and in some cases, spores - are multi-nucleate. During genetic transformation of these spores or mycelia, only one nucleus generally receives the transferred T-DNA generating heterokaryotic colonies. Characterization of genetic changes, such as the effects of gene disruption in the transformants, requires purified homokaryotic lines. Hyphal tip transfer has conventionally been used to isolate homokaryons. We developed an alternative method for purification of fungal homokaryons from transformed heterokaryotic lines of Sclerotinia sclerotiorum. Ultrasound pulses were used to generate bi-septate, unicellular hyphal fragments that regenerate under selection to produce homokaryotic lines that can be easily identified using colony PCR. This technique facilitates the purification of transformed lines, which allows for routine genome manipulation, and should be adaptable for other filamentous fungi.