Would it be an option to start transformation with homokaryons in the first place?

Dear Trung, first You need to know some about the number of nuclei in spores of Mucor circinelloides. If they are uninucleate then You may prepare a very diluted (dependent on the sporulation rate of the fungus You know it better than me) spore suspension with proper concentration, and distribute an aliquot of that spore suspension evenly onto an agar medium amended with the selective agent in accordance with the selective marker applied. To be sure, You may add a layer of such a selective agent-containing agar-containing medium onto the plate after even distribution of the spore suspension aliquot in order to sandwich the spores. This ensures You that if there is germination and growth of your fungus, it is surely resistant to that selective agent applied- if Your materials are active and other conditions (concentration) are well adjusted. For this You shall add the second cover layer when its temperature does not burning the backside of your hand and the medium is still liquid. Sometime mycelia and spores are of different tolerances of freeze-thaw treatments and if mycelia are disrupted easily, You can then use such a treatment to omit coenocytic (aseptate orwithout transverse walls) heterozygous multinucleate mycelia. You may have access to better facilities or materials that differentially target and kill mycelia while spores survive. If spores are not uninucleate, there will be a problem.

Hi Trung. As Babak has indicated, generating single spore isolates is generally the best way to get homokaryotic transformants of a fungus. I would add that one should actually check (microscopically) that small colonies are arising from single spores, since the spores of many fungi do like to adhere to each other. This can often be checked with an ordinary microscope and the 10x magnification, placing the Petri dish (unopened) directly on the stage (inverted or upside right, depending on the agar depth and required focal plane).

However, Mucor is a coenocytic zygomycete and its spores are expected to be multinucleate. This means that even using selective medium you cannot be certain that the isolate you are working with is homokaryotic. The transformed nuclei in the spore will allow the spore to germinate and grow, even if there are also non-transformed nuclei present. As long as there are transformed nuclei present, it doesn't matter how many times you subculture and isolate spores on selective medium, the non-recombinant nuclei can be carried along. If I remember correctly, nuclei are able to migrate within the Mucor hyphae further allowing the non-recombinant nuclei to continue within the mycelium, in spite of your selective pressure.

I do not have a good solution to this problem. You could consider isolating from protoplasts, if you can make uninucleate protoplasts with Mucor. With some fungi, most protoplasts will have only one nucleus, but a small percent will have 2 or more. Thus you would need to confirm that your protoplasts mostly have only one nucleus (fluorescent staining) and then you would need to isolate several colonies growing from single protoplasts in order to get one or more which would be homokaryotic. This is clearly even more work than isolating from spores. If you have access to a FACS machine which can sort cells, you could potentially use this to obtain uninucleate protoplasts.

@ dear Marilyn, may You please introduce the methodology of uninucleate protoplast production. thanks very much.

Hi all, I found that the singer instruments sporeplay can be a very powerful tool for isolation of single spores in an effort to produce homokaryon.

Dear Nir, may You kindly describe the advantages of singer instrument sporeplay compared to normal microscope. I had not heard of such an instrument and after your introduction of that, I searched for its photo. It seems like a microscope with a rail. Thanks.

The sporeplay allows you to manipulate single spores with its specialized needles and transfer them to an organized matrix.

http://www.singerinstruments.com/index.php?option=com_content&task=view&id=310&Itemid=1064

Hi Babak (et al.)

The number of nuclei per protoplast will depend on the state of mycelium you start with, the size of protoplast generated, etc. I don't know whether single nucleus protoplasts of Mucor species (or other zygomycetes) can be generated, but for most ascomyetes the majority of protoplasts should have only one nucleus. Using young mycelia, or just germinated spores, is probably a good starting point for any species, but the nuclear number must be determined by staining (e.g. with DAPI, Hoechst solution or other nuclear stain). If you work with a species which produces uninucleate spores, or multinucleate spores in which all nuclei were derived from the same parent nucleus (e.g. Fusarium), then it is easier to work with the spores than with protoplasts.

Kind regards,

Marilyn

Hi dear Marilyn

Thank You very much for your kind leading. May I ask You a more question? I have transformed T. asperelloides with an peptide encoding gene that should have be secreted because of the signal peptide in its N-terminus, however, the expression of the peptide led to the sterility of the transformant. It apparently produces conidiophores but without spore. As the peptide includes 4 disulfidic bridges then it is litraturally possible because of secretory stress. Now, the question is if we can overcome this sterility through induction or facilitation of secretion with exogenous compounds. If we could do this, we will be able not only to get homokaryotic transformants, but also to get transformants of more biosafety for applications as a biological control agent in field conditions.

Thanks, Best Regards, babak

Hi Babak,

If this is a Trichoderma species, the first thing I would do would be to try cultivating the strain on vegetable juice agar. For example 20% V8 or carrot or perhaps carrot mixed with tomato juice with 1.5% agar. You may have to autoclave the juice and the agar separately (e.g. at double concentration) and then mix them after they have been sterilised, but before the agar solidifies. The acids in the vegetable juice can break down the agar if they are autoclaved together, but it is fine to mix them afterwards, even while they are both still quite hot. I can't guarantee that this will work, but it often does. The choice of juice depends on what is available. Vegetable juice mixtures which contain onion or garlic are not as good for the fungus, so avoid those. The original 'recipe' calls for V8 juice, but this is not available in Finland and I think the carrot worked as a substitute.

Another option is to replace any ammonium in your medium with glutamine. But try the juice first. Be sure to give the cultures adequate light.

It is also possible that your integration has disrupted a gene critical to spore formation, but hopefully it has only disrupted the regulation and not the actual process.

Good luck,

Marilyn

Thanks dear Nir and dear Marilyn for your kind replies.

Thanks a lot for everyone for your kind suggestions.

I have tried using the filter with the pore-size is 5 micro meter to select the small spores (these spores could have one single nucleus).

However, the percentage of positive transformant nuclei was significantly reduced after filtering, and I also could not isolate the homokaryon strains.

No, I am trying to isolate homokaryon strains by subculture without filter. I will check the results after 9th vegetative cycle.