Seed-sterilization of Rhododendron wardii for micropropagation

Success in tissue culture of all plant species depends on the removal of exogenous and endogenous contaminating microorganisms. Rhododendron wardii is a parent of many of the best yellow hybrids and it has been identified as a Least Concern spp. An efficient protocol was developed for sterilization of seeds of R. wardii for in vitro cultivation for mass propagation and conservation. Three sterilizing agents, namely, HgCl2 (w/v), NaOCl (v/v) + Tween20 and H2O2 (v/v) at varying their concentration and time of exposure were tested. Treatment with 25% H2O2 for 20 min was the most effective with no contaminated cultures, where the germination percentage was 92.6% (ex vitro germination percentage was 58.61%) and produced 100% healthy plants. Even though the exposure to 15% NaOCl + Tween 20 for 20 min produced 100% contamination-free cultures, the percentage seed germination was 51%. Seeds could be sterilized with 5% NaOCl, for 20 min and 15% for 10 min for maximum germination (64%), but contamination rate was 1:2. Sterilization with HgCl2 did not produce any satisfactory result (maximum germination percentage = 18%).