Analysis of Relative DNA Content of Termitomyces Protoplast using Propidium Iodide

Abstract

Towards establishment of biodiversity character via analysis of relative DNA content of wild edible mushroom, Termitomyces species is used here for the first time. Dye propidium iodide (PI), an intercalating fluorescent molecule with molecular mass of 668.4 Da is used for staining of Termitomyces protoplasts to analyse the cellular DNA content using flow cytometer. PI binds the cellular nucleic acids at 535 nm excitation and 617 nm emission range. The protoplasts were isolated from liquid MYG grown vegetative tissues of edible mushroom strain Termitomyces heimii. The isolated protoplasts were purified with 0.6M manitol (MW 3350) followed by washing in PBS buffer. Protoplast pellet was suspended in PI (l mg/ml) hypotonic solution and final volume made up with sterile water. Incubation was done at 37°C for 30 min followed by analyzing using flow cytometer. Results showed that staining depends upon the protoplast purity, size and viability. The vegetative tissue showed a heterogeneous protoplast population in terms of sizes which is ultimately reflected on the relative amount of DNA content. The heterogeneous protoplast population in the suspension through cytometry score results the unequal amount of DNA content in every cell meaning unequal size, age and viability. It is established that PI can be used for staining of mushroom protoplast in biodiversity and genetic research.