Hi Massakib

Based on my experience, HgCl2 is the best to use (at least for me!), although you should care about disposing the used solution and the washing solutions in an environmentally safe way. I used 0.1% solution in water supplemented with 0.5% tween 20 (for 10 min) to sterilize clover seeds with excellent success. I also used 0.20-0.30% (for 20 min) solution to sterilize the dehusked rice grains with comparable success. An important practice is to wash the explant after exposure to the mercury for about 50 min in a plenty of water to make sure that no mercury is left in the tissues.

Good luck

I am agree with Dr. gaber but sometime some plant not work this protocol.You try with different concentration and time period change based upon the you selected plant species you work and particular tissue, organ and cell depended. It is required the standardization in the particular plant species. You also try with sodium hypochloride(0.1 - 1%), 70% alochol and  tween20 etc. GOOD LUCK    

Dear Massakib,

Ok thank you Mr. Kalpesh Ishnava and Mr. Gaber M Abogadallah  I will try 

you need to standardize the sterilization protocol. commonly used sterilant is mercuric chloride, sodium hypochlorite, 70% ethanol, antibiotics, antiseptic agents and others. these can be used either alone or in combination. the concentration to be used varies according to the explant used. generally leaves and embryos tolerate mild sterilant and roots, nodal explants need comparatively high strength sterliant. you can also refer few of my articles. luk

I frequently use 10% domestos bleach. Leaf needs 5 min for three times. The corms need 10min for three times. 

Thank you Dr.Sharad Vats  and Dr. Zita Demeter 

Hi, you can just use hypochloryte (2%) for 7 - 10 min. Shake your  submerged material at solution. Don't sterilize too much tissue once, it would raise the contamination chances. Wash with sterile water three times.

It worked for Eucalyptus seeds and axilar buds.

Good Luck.

10 % of commercial bleach where active halogen concentration is around 0.4% is ideal or for such tissue you can also try 0.02% mercuric chloride

I wouldn't use a unified protocol for any of these cases - the type of sterilizant(s), their concentration and time of exposure should be optimized in terms of the particular species, initiation explant (kind of tissue, seeds, bulbs, roots). My preferred sterilizants are ethanol, commercial bleach (frequently Domestos, due to its surfactant content), some fungicides in certain cases, and I avoid using HgCl2 because of its toxicity.

I am agree with Dr. Kalpesh when He said that You have to standard a specific protocol in the particular plant species. We used ethanol and sodium hypochloride with sucess.

But these disinfectants (ethanol or sodium hypochloride) can damage fragile samples especially embryos?

I used sodium hypochloride 2 and 5 min a, and after embryo cultures, they  changed their colors from white to brown 

 Dear Massakib, this is just a matter of adjusting the factors time and concentration. In my PhD I used both substances in the disinfestation protocol of immature zygotic embryos of Bixa orellana and I had no problem. Calcium hypochloride is less harmful to tissues. And certainly HgCl2 is so less harmful than both before

You may use HgCl2 combined with Claforam. You have to establish the proper concentration of the antibiotic to avoid any contamination with bacteria.

Regards

If you can, start with checking out the right concentrations and disinfection solutions in publications which have demonstrated successful sterilization of the particular tissue of the particular plant species you are doing (or at least a particular tissue). Some tissues such as meristem may be more sensitive to a concentration of a disinfection solution than the others such as leaves. You don't want to 'cook' those explants in a disinfection solution before use them for tissue culture.

your question is much too unspecific.

For e.g.: leaves...there are delicate leaves and those with a strong waxy cuticle.

Or embryos. I would start to sterilize the fruit or so first and from there to cut out the embryo. Thus you can use a stronger sterilisation that would not hurt the inner tissue.

I use a standard procedure for disinfestation of explants of any species, for plant tissue culture , which is a 0.5% sodium hypochlorite solution, with a drop of Tween 20 per 100 ml of solution, for 15 minutes, followed by rinsing three times with autoclaved water. When this does not work , it is because the microorganism has penetrated and solving the problem is with other measures.

Thank you all of you :)

Hi Massakib,

By the way, I like your picture. Is it an ocean creature? What is its name?

Delicate tissue can be sterilize by serial dilution method and final dipping in 70% EtOH. However for leaf explant serial sterilization by NaOCl with Tween 20, HgCl2, 70% EtOH and finally by  autoclaved ddH20 is most effectieve

Thank yu @Gaurab Gogoi 

@Yuan-Yeu Yau  : yes it's an ocean creature who can regenerate every damaged part of his body his name Axolotl 

Thanks for the info....interesting......It is an eye-catching animal......Axolotl!!

Do you really need to sterilize the embryo - i found that sterilising seeds and excising out embryos under asceptic conditions is good enough.

Hi anyone knows how to decontaminate in vitro culture plants ?I tried several methods but didn't work . the bleach was too powerful for in vitro plantlets.

It depends on the type of explant u use. Mercuric chloride is used frequently in most of the explants with high efficiency. But there are other sterilizing agents like calcium chloride, bromine water etc which are also effective.

What is the best invitro rooting protocol, of the indigenous plants specifically Eulophia Streptopetala Lindl during tissue culture propagation