Modern plant breeding studies have focused on the production of F1 hybrid varieties which are tolerant to abiotic stress arising from global climate change. Sweet watermelon (Citrullus lanatus var. lanatus) is susceptible to drought stress, and citron watermelon has unique characteristics, which makes it a potential rootstock for watermelon grafting in water-scarce areas. The present study was conducted to assess whether the production of pure citron lines via anther and ovary culture techniques can improve new F1 hybrid rootstock citron varieties in a short time. However, to date, no successful protocol for anther and ovary culture in citron watermelon has been reported. Therefore, the current study was carried out to shed light on the efficiency of growing media composition in callogenesis, embryogenesis, and plantlet initiation in eleven citron watermelon lines. The combination of plant growth regulator (PGR) and genotype induced varying androgenic and gynogenic responses in terms of the percentages of the induction of callogenesis and embryogenesis. The greatest androgenic and gynogenic potential was observed with the combination of MS + 2 mg/L 2.4-D + 2 mg/L 6-Benzylaminopurine (BAP) + 1 mg/L 1-Naphthaleneacetic acid (NAA) + 30 g/L sucrose (IM1 media) in CW21 and CW4 line, respectively. In total, 11 plants via anther culture and 19 plants via ovary culture were recovered and rooted on the solid MS medium supplemented with 0.4 mg/L indole-3-butyric acid (IBA). The ploidy analysis revealed that all plants were diploid (2n = 22).