Upland cotton, Gossypium hirsutum, originated in the Americas and has become the primary cotton species of commerce in the world.  G. arboreum is a domesticated Asian species that is a potential source of genes for upland cotton improvement.  However, upland cotton is reproductively isolated from G. arboreum via post-zygotic breeding barriers.  Recent literature on somatic embryogenesis of cotton suggests a number of media modifications that might also prove useful for ovule rescue of interspecific crosses.  Additionally, endogenous microbes are common in field grown cotton and these potential contaminants must be controlled if interspecific progeny are to be obtained via large scale field crossing followed by ovule or embryo culture.  This study compared nine tissue culture media and two antimicrobial overlay treatments in a factorial design.  The overlay treatments were: a 2 ml overlay of 250 mg/l cefotaxime, 50 mg/l tetracycline HCl, 2.5 mg/l amphotericin B and 50 mg/l benomyl applied when the ovules were plated, and no overlay.  All of the media in the factorial also contained 250 mg/l cefotaxime.  An additional treatment of one medium with no cefotaxime and no overlay was evaluated as a control.  During July and August 2005 crosses were made in a field at Stoneville, MS between the upland cultivar DeltaPine 90 and the G. arboreum accession A2-190.  Fruit were harvested 5 d after pollination, washed with soap and water, then surface sterilized in a laminar flow hood by immersion in an aqueous solution of 2.6% sodium hypochlorite and 0.1% Tween-20 for 10 min with intermittent shaking, followed by immersion in 100% ethanol for 10 min, and then allowed to air-dry.  This surface sterilization protocol was > 99% effective on greenhouse-grown fruit.  For each fruit, ovules were placed on a single 100 x 25 mm Petri dish containing 25 ml of media.  Cultures were incubated at 30 °C with 12 hr of fluorescent light each day.  All of the 11 non-antimicrobial control plates became contaminated (81.8% bacterial, 18.2% fungal).  In contrast, contamination was observed on only 62.1% of 169 plates with cefotaxime but no overlay (82.7% fungal, 10.6% both fungal and bacterial, 6.7% bacterial), and 5.6% of 54 plates with cefotaxime and an antimicrobial overlay (all bacterial).  Germination was not affected by the overlay nor did overlay treatment interact with media.  Media significantly affected germination.  Of the media studied, the highest frequency of germination was observed for MSB2K (Murashige and Skoog basal medium, Gambourg's B5 vitamins, 1.9 g/l additional KNO₃, 20.0 g/l glucose and 2.2 g l gelrite), which averaged 7 seedlings per fruit.  The addition of 0.5 g/l asparagine and 1 g/l glutamine did not affect the number seedlings.  A filter paper growing surface or the addition of 0.5 mg/l NAA and 0.05mg/l kinetin were disadvantageous.