Effect of various sterilization procedures on the in vitro germination of cotton seeds

In vitro manipulations of cotton often require high-quality sterile seedlings as a source of hypocotyl and cotyledon explants for initiation of embryogenic cultures or embryo apexes for shoot production. Unfortunately, in vitro seed germination is often hindered if cotton seeds were collected from the open field and stored under improper conditions. In our case a limited supply of field-grown cotton seeds was received, which necessitated the development of a more effective surface sterilization protocol. Seeds from two accessions, designated as I and II, with very high contamination levels and lowered germination rate were used in this study. These seeds were treated with the most commonly used sterilizing agents, which included commercial bleach, chlorine gas and hydrogen peroxide. Additional steps such as soap-water washes (SW), 70 % ethanol (ETH) and plant preservative mixture rinses were also included in the sterilization procedures to improve the efficiency of tested protocols. Surface sterilized seeds were germinated on Linsmaier and Skoog medium and the percentage of contamination free and well-germinated seeds were recorded for each treatment. Seeds treated with hydrogen peroxide showed significant improvement in germination rate and level of contamination when compared to identical seeds treated with chlorine gas and commercial bleach. The most effective sterilization protocol for all genotypes tested consisted of SW wash followed by ETH rinse and H2O2 sterilization for 7 h. This protocol was successfully used to sterilize seeds of 55 cotton lines.