Background: Monovarietal cultivation of cotton plant allowed the genetic erosion for traditional cotton varieties that proceed essential traits mainly involved in cotton genetic breeding. These varieties need to be preserved for future used. This study aims to evaluate the effect of gibberellic acid on cotton seed germination and the effect of Benzylaminopurin (BAP), Kinetin (KIN), α-naphthalene acetic acid (NAA) and activated charcoal (CA) on cotton seedlings growth obtained from different type of explants. Methodology: The seeds of three improved varieties (KET782, ANG956, OKP768) and five local varieties (Q62, Q64, Q85, Q88, Q92) were germinated with different concentration of gibberellic acid (GA3). Different explants were collected from the germinated plantlets and cultivated in different culture media containing plants growth regulators and activated charcoal in different concentration. Results: We found that Gibberellic Acid activity on cotton seeds germination significantly varied according to the variety and GA3 concentration in the medium. 63.33% germination rate in OKP768 was obtained on the medium containing 0.5 mg/L of GA3 while the medium with 1 mg/l of GA3 gave the highest seed germination in Q85 (75%), Q64 (69.17%), and ANG956 (40.83%). The plantlets regeneration rate varied within the explants in different varieties used. 100%of axillary nodes were regenerated in Q62; Q85 and Q92 varieties while 60% were regenerated with the zygotic embryos in ANG956, and OKP768. Moreover, the medium supplemented with GA3 (1mg/l) + NAA (1 mg/l) induced the greatest number of roots (2.75 roots/plantlet) in the ANG956 and OKP768 varieties. With activated charcoal (10 g/l), ANG956 and OKP768 varieties achieved better performance with respective roots length average of 3.4 cm/plantlet and 2.1 cm/plantlet. The activated charcoal at 10 g/l highly influenced the length of roots with an average of 7.7cm in ANG956 variety. Implication: The protocol established during this study will be useful for in vitro regeneration and conservation for cotton local varieties.