S04 - Session O3 - Methods of asepsis for in vitro establishment and germination of Eugenia uniflora seeds

Authors: John L Griffis *, Malcolm M. Manners

A limited number of scientific studies have been conducted for commercial cultivation of pitanga. Like many fruit crops, clonal propagation of superior selections is highly desirable. However, there are no published studies that have demonstrated a satisfactory micropropagation protocol for this tropical fruit crop. Propagation of improved selections has been limited to grafting, a relatively difficult technique. There are no improved rootstocks available, so even grafted plants may perform differently in the field. To conduct in vitro micropropagation trials, it is often necessary to obtain aseptic material from the recalcitrant seeds and no established protocol for seed asepsis of pitanga is available. Both serious contamination problems and germination failure in tissue culture are not uncommon. This study aimed to define a dependable protocol for surface disinfestation of seeds of Eugenia uniflora for in vitro establishment and germination. Seven protocols were trialed using different rates and/or sequences of disinfestants including 95% ethanol, sodium hypochlorite (NaOCl), hydrogen pyroxide (H 2 O 2 ), and sodium dichloroisocyanurate (NaDCC). The experiment was conducted in a split-plot design in controlled environment growth chambers and repeated. The percentages of both contamination and germination were evaluated at 30 and 60 days after initiation. Vigorously washing fresh seeds in deionized water with Tween 20 for an hour, followed by a I-minute dip in 95% ethanol, then 20 minutes in a 1.5% sodium hypochlorite solution and then 3, 5-minute washes in sterile deionized water was the best of the of methods evaluated. After 60 days in culture, 85% of the seeds disinfested with this procedure did not show any contamination and 70% of the seeds had germinated with both a radicle and a plumule evident.