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This research was aimed to develop culture medium enhancing the germination of self and cross-pollinated seeds in Nymphaea rubra ‘Maeploi’ and Nymphaea pubescens Willd. The self-pollinated seeds of N. rubra ‘Maeploi’ were investigated for surface sterilization method, seed culture medium and light conditions. The self-pollinated seeds were surface sterilization with 20% (v/v) sodium hypochlorite (NaOCl) at various times (5, 10, 15 and 20 min), followed by the second surface sterilization with NaOCl at different concentrations (5, 10, 15 and 20% (v/v)) for 10 min. MS medium at different strengths (MS, 1/2MS, 1/4MS, 1/8MS) was also studied to rescue the cross-pollinated seeds, compared to control. The results showed that the fruit set percentages of N. pubescens x N. rubra ‘Maeploi’ and N. rubra ‘Maeploi’ x N. pubescens were low at 5% and 3.33%, respectively. The N. rubra ‘Maeploi’ self-pollinated seeds were soaked with 20% (v/v) NaOCl for 5 min, followed by the sterilization with 5% (v/v) NaOCl for 10 min showed without the microbial contamination. Moreover, the suitable medium for N. rubra ‘Maeploi’ self-pollinated seeds was 1/8MS semi solid medium under dark condition. The germinated seedlings were developed shoots (60%), immature leaves (51.67%), and roots (48.33%) after 4 weeks of culture. Germination percentages of N. rubra ‘Maeploi’ and N. pubescens self-pollinated seeds cultured on 1/8MS were 51.67 higher than those of cross-pollinated seeds of N. pubescens x N. rubra ‘Maeploi’ and N. rubra ‘Maeploi’ x N. pubescens (35 and 31.67%, respectively).
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