Dactylorhiza symbiotic culture

[PLANTMAN1]

I put some Dactylorhiza symbiotic cultures in the fridge in November and have just had a look at them and around 80% of them the protocorms have gone black (so 2 months in the cold). Many look dead. Those given only a month look the same as when I put them in.
Has anyone any experience with them and am I doing something wrong?
The picture is of the cultures in November.

[Neil]

My first thought is how cold is the fridge?  If it is too cold it will kill them.  Simple remedies are to lift the petri dish of-off the glass shelf so it is not in direct contact with it.  Move them to the top of the fridge and as far away from the evaporator coils as possible, ie in the front of the fridge. The coldest I would put them at is 5C so I bought myself a small fridge that allowed me to set the temperature at that. 

[PLANTMAN1]

I wondered that. But I figured that they tolerate frosts in the wild so they should not be adversely affected by cold in a fridge.
I have put most of them in a frost free store in the garden but I am inspecting them every week.
I am not sure what to do with some Spiranthes as they seem to be beginning to shoot without a cold period like Anacamptis protocorms do.

[Neil]

Don't know why it happens but that is how I overcome that issue.

Spiranthes can be treated like Anacamptis for growing on, I just keep them in a frost-free greenhouse under the bench, no need to refrigerate.

[PLANTMAN1]

Thanks for that advice.
Have you had any success with P. bifolia? They seem to germinate at different times so I have various sizes of protocorms. I was a little late securing seeds and probably need to start again in July rather than the end of October.
I have around 15 protocorms that are large enough to shoot, but I have put all of my cultures outside in my frost free store to give them a winter period.
I will be sowing Anacamptis morio, fragrans and papilionaceae in a couple of months (February probably and then again in July to compare if there is a difference in success rates between the two dates) on A36.

Thanks again.

[Neil]

As you are doing it symbiotically, its best to sow them from July onwards then they are in sync with the seasons.  with the wintergreen species, Ophrys, Orchis, Anacamptis etc is best to put them into a jar as the leaves will continue to grow throughout the winter.  Once they have produced a decent size root system, about 20-30mm, they can be transplanted into soil.  Platanthera I found best germinate from immature seeds, and yes they do germinate over a longer time span.

[Anders]

Could it be a fungus effect? I have never seen that kind of browning of asymbiotic Dac seedlings in the fridge, not even after half a year at the back of the fridge. The fungi I grow at work tend to spoil their agar plates rather quickly as they grow. The fungi for  orchid propagation are slow growers compared to ordinary lab isolates, but you also seem to keep them on the same plates for much longer. Just a thought.

Anders

[Neil]

Anders, do you think the fungus has eaten the protocorm? 

[sjusovare]

I would think that the cold temperatures may favorize the fungus over the protocorms in such situations, basically the fungus has the ideal substrate to grow and is not slowed much by slightly above freezing temperatures, while the protocorms have a really reduced activity level...

[Anders]

Hard to tell. The fungus could have eaten the protocorms, or more likely, it may have spoiled the agar by lowering the pH and and releasing all kinds of nasty compounds that are toxic for the protocorms.

Anders

[PLANTMAN1]

Here is what they look like.
The annoying thing is I had 300 of them in the fridge. Ones left for 1 month all look okay (only 200 protocorms). I lost 2 Platantheras too.
Others I put in an under patio storage area are all look fine and have shoots beginning to extend after 4 weeks.

[PLANTMAN1]

I had a query about symbiotics cultures.
I have some Platantheras is soil from protocorms this winter. They were sown in September and around 10 were put into cold storage in an outside building from mid-December to early March. And Then they were placed in jars with B1 fungus and some seemed to develop longer shoots. They were then put into the light for around two weeks as the tips started to go green.
By then but some of them into soil. All of them seem to have done nothing. The protocorms are still there, they have not died. But they also have not grown at all. The pictures show protocorms six weeks apart- and the second image is what they look like now-with a tiny green pimple sticking out!

 I just wondered if anyone had any suggestions what might be happening? Thanks

Peter

[Neil]

I wouldn't worry yet, they do get quite a shock being in the outside world, and temperatures at night are still cold at the moment.  They still have another 10/12 weeks to grow.

[Maggi Young]

Thought this was a good  place to post  about this  new, open access paper on Phylogenomic Relationships of Diploids and the Origins of Allotetraploids in Dactylorhiza (Orchidaceae)
Marie K Brandrud, Juliane Baar, Maria T Lorenzo, Alexander Athanasiadis, Richard M Bateman, Mark W Chase, Mikael Hedrén, Ovidiu Paun.

Systematic Biology, syz035:
 https://doi.org/10.1093/sysbio/syz035