Effective sterilization of initial explants is of vital
importance for the success of in vitro culture of H. selago
shoots. The best results were obtained when the two step
surface sterilization procedure was applied. In the first step
shoots were soaked successively for 1 min in the sequence
of solutions: 0.01 M hydrochloric acid (HCl), 5% sodium
hypochlorite (NaOCl), 2.4 mM citric acid. In the second
step 70% ethyl alcohol (C2H5OH) for 1 min, 5% sodium
hypochlorite in water for 15 min and 7% hydrogen
peroxide (H2O2) for 10 min were used. After this
procedure microbial contamination was not observed in
90% of the plants after 2 weeks of culture. When the first
step was omitted 60% of shoots showed no signs of
microbial contamination after 2 weeks culture. However,
in the second variant when 7% H2O2 was used for 15 min
the signs of over sterilization were observed after 1 month
of culture. Treatment with 7% H2O2 solution for 15 min
produced death of otherwise fairly resistant H. selago
explants.
After the second week of culture signs of endophytic
contamination were observed. Bacteria and fungi developed
at the base of the green shoots. Appearance of the microbial
colonies was similar in all of the cultures