Successful plant transformation depends on regeneration of plants from transformed cells. The current commercial transgenic cottons were derived from the tetraploid Coker genotypes. This lack of variability in transgenic cotton could potentially contribute to a narrow genetic base. Therefore, there is a need to broaden the number of regenerable cotton lines. Through a combination of technique, media, and timely manipulations, we have developed a method to produce large numbers of somatic embryos (SEs) in two tetraploid as well as in two diploid accessions. Callus was initiated from hypocotyl or cotyledon explants and transferred, within a critical time frame, to a callus proliferation/maintenance medium. Potential embryogenic calli were then identified and transferred to liquid culture for four weeks, strained through a mesh screen to enrich for embryogenic cells and placed on an embryo development/maturation medium. Large numbers of somatic embryos were reproducibly developed from all these lines. Mature SEs, placed on medium with no hormones, germinated and produced plants. Efforts were undertaken to improve the plant regeneration efficiency by a combination of media composition from cultures of different age. In addition, genetic changes due to the culturing process (somaclonal variation) were also investigated in a preliminary study. Several methods have been used to assess the degree of somaclonal variability both at the macro- (chromosome analysis) and micro- (RAPDs, AFLPs, RFLPs) molecular level. Preliminary micro-molecular analysis, using fluorescently-labeled AFLP primer pairs, indicated little somaclonal variation due to the culturing process. This report offers, for the first time, a reliable and reproducible method to obtain regenerable and highly embryogenic lines in diploid Gossypium species as well as tetraploid commercial lines that can be utilized for development of efficient genetic transformation systems. In addition, the methodology followed has the potential for improving embryogenicity of other commercial recalcitrant cotton lines.