Induction of haploid embryos and plant regeneration via anther culture in stevia (Stevia rebaudiana Bertoni)

Stevia, a natural sweetener derived from the leaves of Stevia rebaudiana, is gaining popularity for its zero-calorie sweetness and health benefits. Haploid production through anther culture represents a promising technique for advancing genetic improvement and accelerating breeding programs in Stevia rebaudiana. In this study, the effects of seven different media formulations labeled as T1 to T7 were examined, each with specific compositions: T1 (MS), T2 (MS+0.5 mg l-1 NAA + 0.5 mg l-1 BAP), T3 (MS+1.0 mg l-1 NAA + 0.5 mg l-1 BAP), T4 (MS+2.0 mg l-1 NAA + 0.5 mg l-1 BAP), T5 (MS+0.5 mg l-1 NAA + 0.5 mg l-1 Kin), T6 (MS+1.0 mg l-1 NAA + 0.5 mg l-1 Kin), and T7 (MS+2.0 mg l-1 NAA + 0.5 mg l-1 Kin). Additionally, three stevia cultivars such as Levent 93, Turgut 82, and Tütüncü were investigated to determine their effects on the production of embryogenic callus, embryos, and plant regeneration through anther culture, with each experiment conducted three times. Subsequently, shoot multiplication occurred in four media designated as M1 (MS), M2 (MS + 0.25 mg l-1 BAP), M3 (MS + 0.5 mg l-1 BAP), and M4 (MS + 1.0 mg l-1 BAP). Following shoot production, the shoots were rooted in three additional distinct media (1/2 MS, 1/2 MS+1 mg l-1 NAA, and 1/2 MS+1 mg l-1 IBA). Finally, the produced plants were acclimatized, and their ploidy level was determined through chloroplast count, stomata count, stomata size, and chromosome count. The anthers were cultured at the uninucleate stage, characterized by a greenish color, fragility, and two lobes containing numerous microspores. The filaments were completely removed before culture. After culturing, the anthers were kept in the dark for ten days. Following this period, they initiated embryogenic calli in most of the media, except those containing T1 (MS). The anther wall split, allowing the microspores to initiate the formation of embryogenic calli and embryos, and in some cases, they directly developed into plantlets. Based on the results and observations, all cultivars and media responded well. However, there were significant differences among them. In the first experiment, the mean callus ratio, mean embryo number and percentage per callus were higher in Levent 93 compared to the other cultivars. Regarding treatments, the mean callus ratio was higher in T4, followed by T2, T3 and T5. The number and rate of embryo production were higher in T4, T7, T5, and T2 media. The results of the interaction showed that Levent 93 had the highest mean callus production, mean embryo production, and mean root production in T4. In the same treatment, Tütüncü, followed by Turgut 82, showed the subsequent highest callus production. In the second experiment, the mean callus ratio, the average number of embryos and percentage per callus were higher in Levent 93 and Turgut 82 compared to Tütüncü. Similarly, T4 showed a better response in terms of callus production, embryo number, and percentage. There was no significant difference between T4 and T7 in terms of embryo number and percentage. Turgut 82 showed good results in T4 for mean callus production, embryo number, and percentage, followed by Levent 93 in terms of average callus production in the same medium. However, there was no significant difference in embryo number and percentage between Turgut 82 in T4 and Levent 93 in T7, T6, and T4 media. In the third experiment, mean callus production was higher in Levent 93 and Turgut 82 compared to Tütüncü. However, there were no significant differences observed in embryo number and percentage among all cultivars. In terms of average callus production, T4 showed a positive response similar to T2, followed by T3. Nonetheless, in the third experiment, there were no significant differences in embryo production numbers and rates; T4, T2, T5, T6, and T7 showed similar results. When compared to Turgut 82 in T4 and T2, Turgut 82 in T2, Tütüncü in T6, and Levent 93 in T3 exhibited higher average callus production. There were no significant differences in embryo production numbers and rates due to interactions between media and cultivars. There were no significant differences observed in shoot multiplication between cultivars. However, M3 media produced significantly higher shoots compared to other media across all cultivars. Shoots were successfully rooted in 1/2 MS media. However, the rooting of shoots was lower in other media. The half-strength MS medium produced directly root to shoot while hormone-containing MS media produced callus first and later on a bunch of roots. Acclimatization occurred within 20 days. Stomatal analysis identified differences between plants derived from in vitro anther culture and the control plants, revealing the presence of haploid and other ploidy levels among the anther-derived plants. In summary, this study explores the potential of anther culture for haploid plant production in Stevia rebaudiana. Significant variations in callus production, embryo formation, and embryo rate were observed by evaluating seven media formulations and three cultivars. T4 emerged as particularly effective in promoting superior outcomes across multiple parameters. Successful acclimatization and ploidy assessment underscores the practical applications of this biotechnological approach in stevia cultivation. This study has the potential as a resource for future research aimed at optimizing media formulations and ensuring genetic stability to further advance stevia production.