Explants sterilization of Eucalyptus using NaDCC

Sterilising seed with 500ppm NaDCC

by mfdes » Tue Jun 08, 2010 5:47 am

Hi everyone,

I normally disinfest my explants and seed with 5,000 ppm NaDCC, 5-10m for seed, or under 2m for leaf and stalk tissue.
A while ago I was enquiring about using GA3 for recalcitrant seeds. I normally use a 500ppm GA3 solution for 24-48h, followed by the disinfestation protocol above. Someone suggested using a combined solution of 500ppm GA3 AND 500ppm NaDCC for 24h to disinfest my seed.
I know 24h soaks with low concentration disinfestant are used with tissue, but how well do they work on seed? Is it possible to combine GA3 treatment and disinfestation? Will the seeds not imbibe too much NaDCC?
This brings me to the question of, if the imbibed NaDCC is not harmful, why rinse it at all? I have seen it listed as an ingredient in medium used to inhibit microbial growth...

I found NaDCC by accident in the supermarket, with the commercial name of
AQUATABS(TM), developed by Bayer, to be used in disinfection of fruits,
vegetables, water and nursing bottle. It is presented in tablets, whose
contain 167mg of sodium dicholoroisocyanurate.
So, i prepared 1 liter stock solution with 5 tablets. The final
concentration was 0,5% active chlorine, because each tablet has 100mg of
active chlorine (167mg NaDCC)
The medium was prepared diluting (distilled water) 2XMS frozen stock in MS
normal strenght.
I preferred to use Phytagel cause of its clarity to observe contaminants.
pH=5,8. Microwave was used to dissolve gelrite with the amount of medium +
0,01% active chlorine (NaDCC) in the final volume.
After ebullition, it was dispensed in non-sterile glass vessels and wrapped
with alumminium paper. Nothing was sterilized. I forgot the autoclave. It's
ready!!

The inoculation was done under sterile and non-sterile conditions.
Under non-sterile and open-air condition, after the subculture of
aseptically-grown eucalypts explants, the surface of the medium and
explants was sprayed with 0,05%(Cl) solution of the same product.
Up to that time (7 days), no contaminant was observed under both
conditions. I am observing its effect in growth and development of
the plants.
Today, i prepared again this medium, but using agar, and spraying
the vessels, with 70% alcohol, previously.