In general all tissue culture procedures need perfect aseptic conditions right from the inoculation material, media, glassware including laboratory.

You mentioned buds, the explant material should be thoroughly cleaned I mean sterilized as per the protocols then do it carefully certainly you do not get any contamination.

Check the following you get some instructions.

Good Luck!!!

http://www.gbtrp.com/journal/ijbt%20volume%20no%201(1)/ijbtapr1510010104.pdf

http://220.227.138.214:8080/dspace/bitstream/123456789/642/1/Biotechnology+of+curcuma.pdf

Rapid micropropagation of Curcuma longa using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Plant Cell, Tissue and Organ Culture. March 2005, Volume 80, Issue 3, pp 347-351

Thanks a lot i have been trying many protocols still am having contamination. Recently i have been removing the outer leaf sheaths of the rhizomal buds still they are affected in vitro ..

Dear Bibi, try to follow this procedure before the explant sterilization :

Curcuma plants should be transferred to the greenhouse and conditioned (phase 0 of micropropagation process) spraying twice with 0.10 g L-1 Benomyl fungicide every 15 days. The explants should be cut after an additional period of five days without treatments (Mensuali-Sodi et al., 1997). The explants with buds should be washed using water and sodium hypochloride (0,2%) for 12 h changing the solution every 3 hours. Then, you can briefly soak the explant into ethanol 80% and let they dry under cabinet flow for a few minutes. Prepare a solution using PPM 4 % (40 ml/L) + MS/4 pH 4.5 and let the explant soaked in x 4javascript:8 h in the dark.

Sterilization: reduce and refine the explant then, use Calcium hypochloride 7% (prepared from a filtered saturated solution) for 10 min. wash with sterilized water 4 times.

Add some drops of the PPM solution with a Minisart filter over or into the medium .

You can try to operate with the contaminated explants in the same way hoping for a decontamination...

bye and let it know...

Dear mariella i am an undergraduate student and ppm is not available in our uni lab so any alternative for ppm ??? And do u have that published paper with u can u send m a copy of it ??

Hey! PPM is not chemical it stands parts per million. read the following you get clear idea how to make hormone solutions. Mariella Lucchesini clearly mentioned 4%., so weigh 4 mg carefully and make solution.

"This is a way of expressing very dilute concentrations of substances. Just as per cent means out of a hundred, so parts per million or ppm means out of a million. Usually describes the concentration of something in water or soil. One ppm is equivalent to 1 milligram of something per liter of water (mg/l) or 1 milligram of something per kilogram soil (mg/kg)"

thank loads jetty ... i know the meaning of PPM as part per million but in tissue culture it has another meaning as well.. that's why i was asking .. thanks a lot for replying .

Informations on this link: http://www.plantcelltechnology.com/about-ppm/.

PPM is a liquid mixture and 4% stay as 4ml/100ml of the MS/4 solution.

See you for other questions

May be a commercial product "PPM™ (Plant Preservative Mixture)". Come across now. Mariella gave information go through that.

Acronym is "ppm" expressed in lower case (small letters). I answered for that. Sorry for the confusion.

It is best to use plant material that has been developed under controlled conditions, for example in a culture chamber, in a laboratory or in a nebulization chamber and then continue with the sterilization protocols (alcohol, bleach, etc)