Hi, Negin,

have you used proper air exchanges? What is the ratio between key nutrients in VS medium? Which gelling agents have you used?

I suggest to use :

1. more appropriate medium with optimal nutrient ratios.

2. make a good air-ex-change to prevent ethylene accumulations.

3. may be use good agar.

All these details were importnat. Let me know which other conditions have you used: light intensity? Hormones? etc..

Good luck!

I use VS medium that contains 100 mL Macro elements and 100 mL NH4NO3, 10 mL Micro elements, 10 mL Vitamins, 10 mL Ferous+EDTA, 100 mg EDDHA, 30 gr Sucrose, 8gr Agar-Agar(Merk) for 1 litre medium.pH is 5.7 and the tempreture of growing room is 24 and the light intensity is 40 micromol/second. I use foil for the vials to put on top of them.in this picture I use 1 mg/L BA with 0.1 mg/L NAA.I will wait for your comment

Good Luck

Hi, Negin,

I cant find what is VS medium and how much they contain in µM or mM..

I think you have too high nitrogen contents (I assume that you add additional 100 mM NH4NO3, not 100 ml). Fe concentration is very high as well. What was the reason for EDDHA addition? in this conditions you remove many essential cations! Yu should optimize your culture medium to solve the problem..

Hi Taras,

Vs medium is actually Ms medium with additional Fe in EDDHA form.because before this problem we had yellow leaves in other species like R.canina and also this medium is suggested for Roses. This problem was solved for R.canina by adding additional Fe. Also we have these yellow leaves in R.foetida in Ms medium without additional Fe.

Thanks for the information. Have you tried other way to recover leaf beside Fe? In MS medium you can find N:P ratio 40:1, which is too low. In the case of small volume P became a limiting factor very rapidly, and high N may be the big problem. This is one possible reason. Also, I have seen that your agar is too brown. may be you over-autoclave medium? In this case brown color related with sugar karamelization, which also is very bad. Have you tried other medium like B5, or one publish in my paper with more balanced nutrient contents?

Good luck!

Dear Taras,

no I dont try them.can you send for me the ratio of the medium?which Agar I should use?Do you mean I should lower the N concentration of the medium?forexample use half of 100 Mm?

Good luck

Hi, Negin, you can use at least 1/2 N contents from MS, for example, you can replace 1650 mg/L NH4NO3 to 300 mg/l HN4H2PO4,. You should reduce ZnSO4 at last 2.5 times and increasing Cu at least 10 times. Moreover, you can add 200 mg/l casein hydrolisate (Bacto-Tryptone) and 1 mM MES for pH stabilization.

I think these changes will help!

Thanks alot for your answer.Do you know anybody who works on Roses?esp Rosa foetida or R.canina or R.beggeriana?I really want to be successful in my project. I do your suggestion and tell you the results.

Thanks alot

May be authors of this paper?

http://link.springer.com/protocol/10.1007%2F978-1-60327-114-1_16

You can also look for the person who cited this paper.

By the way, alufloie is not an excellent way to cover jars: you can try to use more transparent cover to make air exchanges.

Dear Negin,

May I know, all of you explant colour change to yellowish or only some?

Have you checked the temperature inside of culture room?

Did you check expire date of used agar?

Does the media had solidified properly? You would check by opening one or two containers and test the media or even you can shake them. Solidify media shouldn't change the shap after shaking.

I had t&is experience before and base on the photo your problem is very similar .

Dear Reza,

all of them turn to yelllow.yes the tempreture is 24 centigrade. I dont have problem with the solidity of Agar.what did you do for your problem?

Dear Taras,

I read this paper before. I know about this. I want to change the cover of the vials

Best Regards

You can use this type of jar. This type give you a good aeration. Perforation of the top and insertion of air-transparent material is very important!

Thanks alot Dr Taras. I will tell you the results.

Salam Negin

In Tropical Hibiscus Shoot necrosis is eliminated by replacing FE-EDTA with FE-EDDHA and increasing CaCl2 in medium .Give it a try .I also find that in one protocol MgSO4 , FeEDDHA and Ca (CaCl2 was replaced with Calcium Gluconate because increasing CaCl2 can have negative effectes on shoots because of extra Cl and calcium gluconate is safe, just low in Ca so you need to add much of it http://www.aseanbiotechnology.info/Abstract/21018038.pdf ) are increased to product healthy leafs and shoots .

all of my rose explants show that problem and die and i don't want to try again untill i can make better growth condition . if you can find a RITA bioreactor or similar one it will help too .I do TC in a very difficult condition but there is no problem with agar ! i use food grade agar for all explants Echinacea , Begonia , Lilium species and hybrids , Apple , Carnation (discontinued because of vitrification and low commercial value) and 13 hybrid of Gladiolus are all doing well in my agar ,

I wish you Good Luck

Also, you can repalce CaCl2 by CaNO3. Probably, EDDHA can interact with irion more specifcically, bacause EDTA in solution may also interact with Ca and other divalent cations. In this case aviablity of these cations were reduced.

Dear Negin, I agree with Mr.Taras, maybe u can get response by manipulating media.

As I mentioned before, I experienced this problem but my solving method was different. In one species of rare Orcgid changing to liquid media showed the best response and in one tropical plant, just the shoots came out after media was changed.

I suggest you,apply the different things at the same time and after the best plant reaction following that method. Otherwise, waiting to observe the results one by one, only will waist your time.

All the Best

Dear Negin,

all above especially the offers related to the change in medium formulation seem plausible for yor experiment. You can also try transparent cling film sold in markets. Cover the mouth of the jars using this film to get more light inside. Al foil may create problem !

Do any body suggest S&H medium or B5 medium to solve this problem?

Hi, Negin, B5 and SH mediums are much better to compare with MS. Just look on the ratios between key nutrients on these medium.

Why are they better?

Hi, Negin,

for optimal growth plants need 6 macro and 6 micro-nutrients, in optimal ratios. If some element is in excess, but other is deficient, plants can not perform normal metabolism and have chlorosis, necrosis, leaf senecse (yellow), root system defect etc. It was know since long time ago that N:P:K ratio should be 5:3:1, Cu/Zn ratio 1:2 etc. MS medium is too far from balanced ratios, but it is good for callus growth (exactly for what it design originally)!.

You should also consider autoclaving time and try to minimize volume of the medium per bottle during autoclaving: http://onlinelibrary.wiley.com/doi/10.1111/j.1399-3054.1995.tb00947.x/pdf

Salam Negin

Take a Look at

Multiplication of Rose species http://hortsci.ashspublications.org/content/29/5/431.4.abstract?related-urls=yes&legid=hortsci;29/5/431-c

and this is Rose Multiplication medium that phytotech labs offer in USA

http://www.phytotechlab.com/TechInfo/R757-info.pdf

Good Luck

Dear Taras

I pour 15-20 ml medium in each bottle.and the time of autoclaving is 20 minutes. should I decrease it?

Thank you Mohamad.

I think that 15 minutes is enough, It is important that the medium do not became brown/yellow. It is a good idea about B5 medium, you will have better results!

Dr. Paternak is right. MS medium is used widely. But, it is good at callus growth, and they tried it on a fast growing plants, tobacco. Two years after their investion of MS, they offered Linsmaier and Skoog Medium (LS 1964), minimazing some excess amount of macro and vitaming in same plant. Instead, they obtained well developed plantlets.