We tried to culture pineapple n36 for some months, but still have fungus contamination. Any suggestion for sterilization techniques?
I have worked with other material. Disinfestation process was basically, remove all green tissue of field material and then disinfect in NaOCl solution (3%) for 10 minutes, solution, take a flow chamber to reduce the material and make a second disinfestation with NaOCl (0.5-1%). Important, then the explants incubated for 3 days in darkness
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The explants were thoroughly washed with distilled water for three times. Then, the explants were washed with 70% ethanol (for 2 to 3 min). In the laminar air flow cabinet, the explants were treated with 0.1% HgCl2 with the addition of few drops of Tween-20 for inner surface sterilization (for 4 to 6 min). Finally, the explants were washed with sterile distilled water for several times to remove all the sterilizing agents and then the explants were cut into small pieces by sterile surgical blade ranging in size from 1.0 to 1.5 cm long.
This is the surface sterilization method from one of my research. Hope it can help you a bit.
Hi, previously i work with MD-2, Maspine,Josapine as well as N-36. Here are the sterilization procedure that i optimized.
1. remove all the leaves from the crown/sucker until leaving behind a couple of leaves.
2. excised the explant carefully into a cube shape of approximately 5mm³, if your explant is from the field,wash throughly to remove all soil attached to the plant.
3.put the explant under running tap-water for 30 minutes/more.
4.transfer your explant into laminar-air flow
inside the laminar air flow
1. immersed your explant with 95% ethanol for 1 minute.
2.carry out sterilization with 60% clorox ( commercial bleach which contained 5% NaOCl) with 2 drops of tween 20 for 20 minutes.
3. repeat step 2 for another 20 minutes using new clorox solution ( without tween 20 this time)
4.after double sterilization process, wash the explant with sterilized distilled water for 3X to remove excess clorox.
4. use a blaze to remove the soften part of the explant.
5.your explant now was ready to be culture into growing media.
6. so far, it works for all my cultivars. The explant responses after 2 weeks and shoots poping out around 1 month. All the best!
p/s: 1.pineapple's crown response faster than sucker.
2. materials from field require a higher concentration of clorox to eliminate contamination.
hi,
you vary the time period for the second sterilization with HgCl2 ,and also try some fungicides ,it will give u better result,and also careful about the HgCl2 treatment it is very corrosive,tissue may die
best wishes
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