As I do understand, you got contamination of bacteria and not fungal.

I think the advice of Sadrollah is good. For example washing after incubation with Agro.spec. it helps. But if it is a bacterium inside the plant you are going to reciproce, then you dont know WHICH of the many antibiotis is helpful. Maybe you try to find out, which bacterium it is first to narrow the options.

If you do not have sufficient callus, you should replicate the treatments again. But, if you have crisis of enough explants (as you mentioned that your experimental plant is endangered one), you can use antibiotics or disinfectant for surface sterilization (if the contamination is not so high) prior to subculturing your contaminated materials. Note that the success of the survival of the plant materials depends on luck!!! I got success in this way several times.

Thank you to answer me

Another question if I may;

Should I put antibiotics in the culture medium? if so, it does not affect the culture medium or growth hormones?

Dear Bekkaye.

For your second question, it will be better to wash your contaminated callus with antibiotics and then subculture in new medium.

As I do understand, you got contamination of bacteria and not fungal.

I think the advice of Sadrollah is good. For example washing after incubation with Agro.spec. it helps. But if it is a bacterium inside the plant you are going to reciproce, then you dont know WHICH of the many antibiotis is helpful. Maybe you try to find out, which bacterium it is first to narrow the options.

Hi, Massakib, regarding second question: antibiotics ma be only a temporary decision and antibiotics work against bacterial contamination. In the case of fungi antibiotics is not effective. Actually, you can observe callus under the binocular and mas or in-organized growth on the wounding surface. Which king of hystology did you perform? What was the aim of your experiment? Good luck!

Thank you all to answer me

The purpose of my experience is the regeneration of a plant (tree) endangered,

  which does not regenerate naturally.

I haven't had the opportunity to histological studies to confirm if this "mass" is a callus or not.

 After seen that the samples were contaminated, so I decided to redo the experiance.

I wanted to know if the result was a callus, so I redid the same experiance with the same protocol, and if I can add the antibiotic directly into the culture medium (for prevents the development of bateries)? and how about the fungus?

PS: In my first experience I haven't worked under a laminar flow hood, I worked with benzene beak.

Thank you

Hi, Massakib, benzene beak is not an optimal way. You should try sterile bench! For the contamination,there are a specail preservative for this. You can find these products on eb-site of the company. If you aim to get plants. you do not need to do histology. You have to got fresh friable callus and try to get plant regeneration by testing different medium/hormine combinations. Hystology you need only in the case if you want to study mechanism of plant regeneration form your tree. Good luck!

Ok thank you so much for the advice

If you have massive growth of cells/tissue, it is callus. You can take a fresh sample and look it under the microscope to confirm; plant cells are much bigger than bacteria, and bacteria in general have flagellum and move. But it doesn't mean that the callus will differentiate and regenerate plants! You have to work on desinfesting your samples first, it is better than using antibiotics in the media. Good luck!

I would suggest you redo your experiment. If you have contamination in your pilot experiment, the best way is just throw it. For "the mass", if you can't see any phenominon of a shoot, it seems is callus. You can check the soybean cotyledon-nod regeneration/ transformation papers. You can compare the picture in it. Like Paula Olhoft 2003 paper, Planta (2003) 216: 723–735.

if you are going to use antibiotics it is recommended to find out what type of contaminants first so you can choose the right antibiotics. But before using antibiotics you can try surface desinfection using commercial bleach or sodium hypochloride solution.

You can exam the embryo under microscope.

For removing the contamination ,first u try the fumication of the inoculation room.before starting inoculation wipe off all the work place with 70% alcohol and also spraying near work place with the same can reduce contamination upto some extend.try to maintain sterility in term of glassware,spatula forcep , lab coat etc.If possible only one person work at one time in the hood.

try two time surface sterilization by sodium hypochloride,then 0.1%mercuric chloride, for two times before final washing.you have to try sterile filter paper for removing the excess moisture before inoculation.sometimes 20mg/100ml streptomycin is beneficial to remove bacterial contamination .

For regeneration purpose,most of the time compact is suitable.so try to get that type of callus simultaneously in many media with different formulation of growth hormones i.e cytokinin and auxin ratio,and to avoid wastage of media, try small test tube for initiation of callus.try all parts simultaneously.

Did you use immature zygotic embryo for in vitro culture of embryo?

In fact, you could see better by inverted microscopy.

Do you have it?

Regards,

Totik

If you use cotyledon, it should form callus at the excised section. The mass callus can be view as proliferated cells with some characteristics such as isodiamteric, globular type. Some of callus have certain colour to show its embryogenic. You have to decontaminated the environment to make sure the aseptic technique enough. Adding the antibiotic is unadvised. Usually 2,4-D and combination with other auxin promote the callus formation, however it depend on plant species. You can try different PGR combination.Usually i only use 70% ethanol, discard it and submerged the explant with 100% Chlorox(bleach) for 25min to surface sterilize the explants.

Compared with natural tissues like cotyledon and embryo, the newly produced callus is irregular and incmpact, just like this:

http://ejournal.sinica.edu.tw/bbas/content/2003/4/bot444-01.html

Well if you have contamination before going into more steps ahead, eliminate the contamination problem. This could be done either by improving the process of surface sterilization or by adding antibiotics or fungicide to the medium. Any way the type and concentration of both antibiotics and fungicides are important and carefully selected.

As you already have contaminated cultures just observe under microscope and see if the cells are grown. Basically plant cells are bigger than bacteria and you easily can identify fungi due to filamentous nature. If cells are grown out from the tissue that could be considered as initiation of callu

Cut the mass into very small pieces (a few square millimeters). Put some on growth medium with auxin (or a high auxin:cytokinin ratio), others on growth medium with cytokinin (or a high cytokinin:auxin ration). If it behaves like tobacco, you should see shoot development at high [auxin] and root development at high [cytokinin].

Thank you to you all for taking the time to answer me

@Totik Mariani : I used immature and mature zygotic embryo and cotyledon for in vitro culture of embryo

@Alina Wagiran : I used the same disinfection protocol.

@ Wtpsk Senarath : what is the antibiotic and fungicides you recommend me to use?

Well we usually use RAN (A mixture of rifamycine, amoxyline and neomycine) and benlate or thiram but all depend on your causative organism

Ok thank you alot, and I will redo the experience and apply all your advice.

My experience is that - whether there is a callus or not, it comes out after 2-3 subcultures, when the culture becomes stable. Of course this needs an axenic culture, it needs some luck as well to get a contamination-free explant after repeated trials of surface sterilization.

Possibly yes or no. I want to know what good it will do with such a contaminated cell mass.

Firs you have elimnate the contamination problem. Then I prefer use benomyl before surface sterilization (alcohol 70%, 20% commercial bleach for 30' and sterile destilled water). Once you have decontaminated cultures after days or week of inoculated, you should be observe under microscope the cells grown on the excesion section.

sometimes,endophyte may exerts a problem so to avoid that condition ,separate the big piece of callus in to small one by using two sterile forceps gently instead of using scalpel aseptically. This may reduce the contamination of endophytic bacteria.

all the best .

very difficult to answer to. Of course it could be a very hard and slow growing callus, but to say the truth, the pictures are too small. Sorry.

Maybe with all the good answers you got, you start afresh with a small amount. Could you use unripe embryos? Thus you could use a strong outer sterilisation for the fruit, its from a tree, is it?

yes I worked with mature and immature embryos, and these cell formations have appeared after 12 weeks, on mature embryo and on cotyledons,

but on immature embryos is very unclear because of the color of my culture medium which was very dense.

Why is your medium dense? Do you use gelrite? and maybe less of it to have a soft medium.You use tubes or small petridishes? Even this can make a difference. In the beginning I use always petridishes. Less air above in order maybe to prevent oxidation. and browning. And for the immature embryo you start on medium to let it grow into an embryo, I think to use GA. Sorry, I need to check it first, did it years ago. And only then tp apply the medium to initiate callus.

My culture medium is dense because of the agar, and I worked with tubes of cultures, so it is difficult to take pictures.

I think you have to wash the assumed callus antibiotics and then subculture to fresh media.I t is also important to comment that for precision on the type of antibiotics to use, you could for microbiological analysis first.