Identification of Best Surface Sterilization Treatment and Control of Endophytic Bacterial Contamination in Annona squamosa L.

Surface sterilization is most important step in plant tissue culture protocol. In the present investigation, an attempt was made to eliminate microbial and fungal contaminants from the surface and interior of plant material, thus obtaining axenic culture with highest survival rate. Sequential surface sterilizations of hypocotyl, leaf, shoot tip and mature node were carried out to investigate its effectiveness in controlling surface contamination with satisfactory survival of explants. Combination of different surfactant were used for surface sterilization treatments. The least contamination was obtained when hypocotyl explants were treated with 200 ppm cefotaxime and 500 ppm carbendazim along with 0.1% HgCl2 with best survival percentage. Treatments consisting of alcohol treatment, carbendazim (2000 ppm) followed by 1000 ppm cefotaxime, 500 ppm kanamycin, 2% sodium hypochloride and 0.1% HgCl2 sequentially resulted in complete elimination of surface contaminants from shoot tip, soft node and hard node obtained from field grown mature tree. Optimal elimination of bioburden from young leaf (77.38%) were obtained using 1000 ppm carbendazim, 500 ppm cefotaxime, 500 ppm kanamycin and 0.1% HgCl2. Gentamicin used in the medium was able to control the endophytic bacterial bioburden completely in the first cycle of 15 days itself at higher concentration of 96 mol/l to remove endophytic bacterial contamination with out effecting plant growth.