Effect of Different Factors on Androgenesis Induction in Anther Culture of African Marigold (Tagetes erecta) and French Marigold (Tagetes patula)

Introduction Given the economic importance of growing flowers and plants in the world, the use of new technologies and methods in the improvement of ornamental plants in order to market them can play a significant role in marketing of these products and their trade in the international markets. Marigold (Tagetes spp.), is an annual and essential plant that is cultivated as an ornamental plant in many parts of the world (Neher, 1968). But in recent years, marigolds have been used as a commercial source of essential oils, biological compounds, pigments and cosmetics (Anonymous, 1976), control of malaria mosquitoes (Wanzala and Ogoma, 2013), antihypertensive, anti-inflammatory, antidepressant, antimicrobial, antimicrobial (Senatore and Feo, 1999), and control of nematode (Prasad et al., 1992). Anther or microspore culture is known to be an effective method for producing haploid plants (Henry and De Baizer, 1980). Hybrid seed production requires pure line (as a parent), and the double haploid method can reduce the production period of pure lines to 5-6 years. Production of hybrid seeds in this valuable plant is of great importance, and the double haploid method can be important in this regard.   Materials and Methods In the anther culture of marigold, a culture medium containing 0.2 mg/l of Naphthalene acetic acid and 1 mg /l of 6-Benzylaminopurine was used. In this study, the effect of genotype, bud size and mannitol on androgenesis induction of marigold anthers was evaluated during two separate experiments. A factorial experiment was conducted in a completely randomized design with two factors including genotype at 5 levels (T1, T2, T3, T4, T5) and bud size at three levels (3-5, 5-10 and 10-15 mm) for first experiment. The second experiment was also performed as a factorial in a completely randomized design with 3 replications. In the recent experiment, the first factor included the method of mannitol application and the second factor included the different concentrations of mannitol (0.1, 0.2, 0.3, 0.4 and 0.5 M). The factor of application method was in 2 levels: 1: adding different concentrations of mannitol to the solid culture medium and 2: pre-treating the anthers with the liquid culture medium containing different concentrations of mannitol for 24 hours. The T4 genotype was used in the second experiment.   Results In the first experiment, the effect of different bud sizes and 5 different genotypes on callus formation, mean number of shoot per anther, mean number of shoot per callus and percentage of complete plant regeneration in anther culture of marigold were studied. The results of this experiment showed that buds with the length of 5-10 mm have anthers which their microspores are at the proper growth and development stage for callogenesis and shoot production. The T3 and T4 genotypes, (both of them belonging to French species, Tagetes patula), produced the highest percentage of plant regeneration among the various cultivars. In the second experiment, we explored the impact of mannitol treatment on androgenic traits in marigold anther culture. Specifically, we examined two concentrations: 0.1 M and 0.2 M mannitol, both applied in the form of solid culture medium. Additionally, we investigated two concentrations, 0.0 M and 0.2 M mannitol, when applied as a pre-treatment in a liquid medium containing mannitol. These treatments yielded the highest percentage of callus formation. While the pre-treatment of anthers with a liquid culture medium containing 0.5 M mannitol led to the highest mean number of shoot per anther and the mean number of shoots per callus. Also, the pre-treatment with liquid medium containing 0.2 M mannitol showed the highest percentage of complete plant regeneration.   Conclusion Results showed that in marigold, buds with the size of 5-10 mm contained microspores with mid-uninucleate stage to early bi-nucleate stage showed the highest response to the induction of androgenesis. Also, T3 and T4 genotypes belong to the French species showed the highest response to the regeneration. In another experiment, the pre-treatment of anthers with 0.2 and 0.5 M mannitol by using mannitol in a liquid culture medium for 24 hours, respectively showed the highest percentage of complete plant regeneration and the highest mean number of shoot per callus and anther. Chromosome counting results showed that 3 out of 5 examined plants were dihaploid and had 24 chromosomes in their root tip cells, while examined mother plants were tetraploid and showed 48 chromosomes in their root tip cells.