Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp

Area of Science:

• Microbiology
• Cell Biology

Background:

• Protoplast fusion is crucial for genetic manipulation in Trichoderma spp.
• Effective vital staining is needed to identify and select fused cells (heterokaryons).

Purpose of the Study:

• To evaluate fluorescent vital stains for complementary staining of Trichoderma protoplasts.
• To identify optimal stains for selecting heterokaryons after protoplast fusion.

Main Methods:

• Evaluation of several fluorescent vital stains, including tetramethyl rhodamine isothiocyanate, fluorescein isothiocyanate, fluorescein diacetate, FluoroBora T, acriflavine, Nile red, Rhodamine 6G, and hydroethidine.
• Assessment of staining efficiency, stability during fusion, photobleaching, and toxicity to protoplasts.
• Testing of Rhodamine 6G and hydroethidine on multiple Trichoderma strains.

Main Results:

• Rhodamine 6G and hydroethidine stained all protoplasts effectively and remained stable during fusion and illumination.
• Hydroethidine showed potential nuclear staining but was toxic at higher concentrations or upon light exposure; toxicity was mitigated by using minimum concentrations in darkness.
• Other evaluated stains (tetramethyl rhodamine isothiocyanate, fluorescein isothiocyanate, fluorescein diacetate, FluoroBora T, acriflavine, Nile red) had limitations such as poor staining, rapid fading, or loss of stain during fusion.

Conclusions:

• Rhodamine 6G and hydroethidine serve as useful, complementary vital stains for Trichoderma protoplasts.
• These stains facilitate the visualization of fusion frequency and type (dicaryotic, multicaryotic), aiding in heterokaryon selection.