FORUM IN VITRO

Fresh seeds of drosera ascendens were sown on a media 1/3MS+vitamins (B8 missing), sugar 30g/l, agar 6g/l and initial pH=5.60 (citric acid).
Sprouting began in 10 days (plants in the pic are week old):



Later they grew on quite slowly, until they were about 35 days old, then they started to shut down one after another and 40 days old plants looked like this:



First, I thought that it may have happened because of overheating of the area I grew them in, but they continued on dying under colder conditions as well. So the rapid die-out might have occurred due to media depletion(?) I managed to save 5 plants by deflasking, we'll see if they catch on or croak. Will report later:-)

Hi Dusan,
this does look like something is toxic in your medium. I dont know D.ascendens, but 20mM of nitrogen ( 6,6mM of NH4 ) can be too high.Also check your micronutrients ( KI, CuSO4 can be pretty toxic to CP )

Thanx Milos!
I also think next time I will cut down the content of NH4NO3 from 550mg/l to about 280mg/l and see what happens. Also I will skip Fe and Cu completely and add extra 150mg of ascorbic acid.

Adding ascorbic acid or other antioxidants to media is not good idea at all. This will only hide sign of toxicity.It is much better to find what is causing the toxicity.Dont skip Fe it is required by plant in pretty high levels. You can try to replace FeSO4 with FeEDTA or FeEDDHA they are more stable and have lower toxicity or simply cut down the FeSO4 level.

I make adjustment pH by the help of HCL and KOH. (citric acid ?)
I attach a photo D ascendens were sown on a media 1/3MS
The problem must be elsewhere.

Michal

Dusan, i remember you have mentioned you are using cheap food agar from store. This could be also problem due impurities -> this is reason why you must lower the pH, because such agar is too alkaline.

Hi Dusan
If I am right , you have used agar from me. Milos said it could be because of it . It is possible , cause it is about 1 year old . I don´t know it´s durability .
But I have to say it was not bought in common shop. I´ve got it from Milos and his material shouldn´t be toxic , should it ?

Hi everybody! Leos, I used another agar, from a foodstore as Milos says. I did not measure much contaminants in my sample, but Milos you did find quite a lot of salts contained in foodstore agar if I remember correctly. When Michal used basically the same medium and his seedlings were fine, I try to find differences in our media since other factors dont play important role (assuming they were about the same as Michal's). I came to a conclusion that there are 2 reasons for the plants' collapse. (One of them or both in synergy):
1. toxic impurities in foodstore agar
2. my medium DID NOT have myo-inositol (B8) in the media, maybe for ascendens it is vital

As for the Fe, Milos, I have plants growing just fine without it, I guess it depends on actual species whether it is needed. I know the best solution would be using FeNaEDTA, but I do not have this chelate form so I use salt form. I cut down on it, used 5ug CuSO4.5H2O and 50ug FeSO4.7H2O. I still did not find a suitable source of inositol so I am skipping on it again.
I got some SIGMA-ALDRICH molecular microbiology-grade AGAR so this time I use this fancy stuff to exclude the possibility od side-contamination. Unfortunately, I cannot try the same experiment with pure agar since I dont have any d. ascendens seeds at the time. I will do so when I get some seeds.
About the pH, when I measured it (after the agar meltdown), it was 5.67 (correlated with temperature). I do not do it after autoclaving since I autoclave it distributed in jars already and this would be risky and time-consuming).
Good news is that all 5 remnant survivors, removed from media are in acclimatization process on the substrate and all 5 are growing very well.

Fe is essential microelement. If the plants are growing, it is probably due small content of Fe in water or agar or other chemical compounds.But 50 ug of FeSO4 is too low, you need about 10-20 mg FeSO4/L or more. If you need i can send you small amount of FeEDDHA.

Uncomplexed Fe is absorbed by plants harder than complex with EDTA, but i don't think that it was source of problem.

Bad-quality agar also shouldn't have so big effect on plants.

Many medias don't have inositol and most plants grow well.

I think that you heat-burned your plantlets I did it with mine so many times...

other possibility [similar symptones]- when i sow nepenthes seeds on 50% MS many germinated seeds died after ~1,5 month, and growth of others was very slow- too high nitrogen concentration "burned" them but in the other way.

Addition of citric acid in my opinion is not good idea..... if you want to buffer medium add MES.

Regards