Dear Kanchan Nene

Wash the prepared plant material in a detergent-water mixture for about 20 minutes,

Transfer the washed plant material to the sterilizing chlorox solution. Shake the mixture for 1 minute and then leave to soak for 10-20 minutes. you can use sodium hypochlorite, several important details, alternatives and concentration in the next article to avoid contamination in each step.

http://www.apsnet.org/edcenter/k-12/teachersguide/plantbiotechnology/documents/planttissueculture.pdf

http://studyread.com/plant-tissue-culture-techniques

http://www.ncbe.reading.ac.uk/ncbe/protocols/planttissue.html

http://ejournal.sinica.edu.tw/bbas/content/2000/2/bot12-08.pdf

http://www.slideshare.net/AliaNajiha1/basic-principles-and-protocol-in-plant-tissue-culture

http://www.sigmaaldrich.com/life-science/molecular-biology/plant-biotechnology/tissue-culture-protocols.html

http://www.springer.com/us/book/9781617798177

http://link.springer.com/book/10.1007%2F978-1-61779-818-4

David E. Evans, Julian O. D. Coleman, Anne Kearns. 2003. Plant Cell Culture.Basics (BIOS Scientific Publishers) Basics from background to bench The BASICS (Garland Science) Series,The Basics. Garland Science,Pp. 194. ISBN185996320X, 9781859963203

Victor M. Loyola-Vargas, Felipe Vázquez-Flota. 2006.Plant Cell Culture ProtocolsMethods in molecular biology. Biomed Protocols Springer Science & Business Media,Pp.393. ISBN1592599591, 9781592599592

B. N. Sathyanarayana.  2007.Plant Tissue Culture: Practices and New Experimental Protocols. I. K. International Pvt Ltd, Pp. 316. ISBN8189866117, 9788189866112.

Regards

Prof. Houda Kawas

Please have a look at these links.

https://www.researchgate.net/post/how_to_prevent_the_microbial_contamination_in_plant_tissue_and_cell_culture

https://www.researchgate.net/post/Fungal_contamination_on_tissue_culture

Thanks alot i will surely refer papers and follow the protocol

Please check these PDF attachments also.

it depends on the material you use as explants. If they are tough enough you may (strong seeds ...) you may start immersing them in dil. acid, then washing for 2 h. under running water, after which you may follow the regular protocol using sodium hypochlorite then d. st. water. 

thanks sir. im working on leaves. though its helpful for my coleague

Generally we optimize the surface sterilization time for each plant/tissue as it differs from one to other, but i think you must have done that. So apart from above suggestions, you can do one thing- give those fungi to some plant pathologist for identification and then he/she can recommend you the correct fungicide.  

Also check whether the plant on which you are working, does not contain any type of endophytic fungi.

All the best

thanks a lot ashutosh sir.

Ex plant under running tap water for 15 min and than add 10 drops detergent for fine 5 min. Again running water for 15 min. This protocol use because all fungal spore adhere the surface of the explant. Even after chemical sterilization in higher concentration not remove form the explant and remain in active. Again when ever favorable temp is available spore is active and grown. It is possible only detergent useful remove the spore from the explants. You try with simple experiment.

you can use Nystatin  (500 mg per liter) to sterilize the surface After that clean the surface with ethanol 70% three times.

You can try double disinfection, with 70% ethanol, sodium hypochloride between 20-50% of  2.7-3.1 active hypochloride.  You can as well use mancozeb solution for 5mim. 

can u send the pics of contamination in the vessel?

Dear Kanchan

Fungal contamination is a common problem during explant initiation in PTC, especially in rainy season due to high fungal activity and growth.

Please try to do follow:

Try to avoid initiation during rainy season. Or

Periodical 2-3 spray of mother plant(s) using fungicide (Bavistin and or Redomyl 1gm/L) to reduce contamination level or at least on those branches, to which u r going to taken as an explant and cover with poly bags, if possible.

After spray schedule, cut the branches and put in antioxidant solution (100mg/L each of Citric acid and ascorbic acid along with 10 drops of Tween 20) and bring to lab. Wash thoroughly in running tap water (20 min) and give treatment of   Bavistin and or Redomyl 1gm/L+ 5 drops of Tween 20 for 30 min, clean explants 2 times with sterile distilled water. Treat with 2% Sodium hypochlorite (20-30min) and or 0.1% HgCl2 (6-15 min); finally 2 wash with distilled water. Explants were blotted dry and initiate in initiation culture medium.

Hope fully its will solve your fungal contamination problem.

Regards

Thank you so much all of you for your timely help. 

I agree with Rajesh, avoid the rainy weather. Preferably grow your plants destined for initiation in a clean greenhouse with regular fungicide sprays and drip irrigation as opposed to overhead irrigation. Ensure they are in the best of health for optimal initiation success.  Wash freshly cut shoots under running water for 10 min, then a bleach solution as described by other researchers, followed by 2 sterile water washes then plate up.  The better the mother plant health, the more likely your initiations will be successful.  All the best,

If you have seeds, I would try to start from seeds. Sterilized the seeds, grow them into small seedlings in medium. Once you have these clean seedlings, you can use their tissues as explants for tissue culture (such as induce callus...or other tissue-culturing experiments).