Effects of Biotin and Ascorbic Acid Applications on Haploid Embryo Induction in Semisolid and Double Layer Nutrient Media in Pepper (Capsicum annuum L.) Anther Culture
Abstract
Generation of homozygous double haploid (DH) lines by androgenesis is a promising alternative to selfpollination programs across generations. Despite the routine use of anther culture in peppers, there are still many bottlenecks and improvements in methodology are required. The aim of this study was to determine the effects of the structure of the nutrient medium (semi-solid and double layer) and the addition of biotin and ascorbic acid to the nutrient media on obtaining haploid embryos by anther culture method. MS (Murashige and Skoog 1962) medium containing 4 mg l-1 NAA, 0.1 mg l-1 BAP, 0.25% activated charcoal, 30 g l-1 sucrose, and 10 mg l-1 AgNO3 (silver nitrate) were used as the basal nutrient medium. A total of 8 nutrient media compounds were studied using 0.05 mg l-1 biotin and 0.5 mg l-1 ascorbic acid separately or together in semi-solid and bi-layer (double-phase) nutrient media. Solidification of nutrient media was achieved with 7 g 1-1 agar. The cultured anthers were subjected to high-temperature pre-treatments at 35 °C in continuous dark conditions for 2 days. Then they were taken to a climate chamber at of 25 °C temperature adjusted to 16/8 hour photoperiod. It has been observed that the success of obtaining embryos of semi-solid medium was higher than double-layer medium. The addition of biotin and ascorbic acid to the nutrient medium provided 8.8 fold increases in embryo regeneration compared to the control medium. In the presence of only one of biotin or ascorbic acid in the nutrient medium, the number of embryos increased compared to the control.
Keywords
Androgenesis, vitamin, breeding, pure line
Supporting Institution
No financial support was received for this study.
Thanks
The authors would like to thank the Faculty of Agriculture, Eskisehir Osmangazi University for providing their greenhouse to cultivation of donor plants
References
- Açıkgöz, N., İlker E., Gökçöl, A. (2004). Biyolojik Araştırmaların Bilgisayarda Değerlendirilmeleri, Ege Üniversitesi Tohum Teknolojisi Uygulama. ve Araştırma Merkezi Yayınları No:2, Ege Üniversitesi Ziraat Fakültesi Ofset Atölyesi, Bornova-İzmir, 202 s (in Turkish).
- Ahmadi, B., Ebrahimzadeh, H. (2020). In vitro androgenesis: spontaneous vs. artifcial genome doubling and characterization of regenerants. Plant Cell Reports. 39:299–316. Doi: https://doi.org/10.1007/s00299-020-02509-z
- Al-Khayri, J.M. (2001). Optimization of biotin and thiamine requirements for somatic embryogenesis of date palm (Phoenix dactylifera L.). In Vitro Cell. & Dev. Biol.-Plant. 37, 453–456. Doi: https://doi.org/10.1007/s11627-001-0079-x
- Becker, M. G., Chan, A., Mao X., Girard, I. J., Lee S., Elhiti M., Stasolla, C., Belmonte, M.F. (2014). Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis. Journal of Experimental Botany. 65(20) 5903–5918. 10.1093/jxb/eru330. Doi: https://doi.org/10.1093/jxb/eru330
- Ceyhan, A.P. and Aktaş, H. (2020). Development of Three Dihaploid Nose and Bell Pepper Lines with Anther Culture Technique. Eurasian J Bio Chem Sci, 3:199-205, Doi: https://doi.org/10.46239/ejbcs.822593
- Cheng, Y., Jiao, Y., Miao, R., Tian, R., Liang Y., Qiao, N. (2020). Exploring differentially expressed genes of microspore embryogenesis under heat stress in sweet pepper. African J. of Biotechnology. 19(9):661-674. Doi: https://doi.org/10.5897/AJB2020.17194
- Çömlekçioğlu N., Ellialtıoğlu, Ş. Ş. (2018). Review on the Research carried out on in vitro Androgenesis of Peppers (Capsicum annuum L.) in Turkey. Review Paper: Research Journal of Biotechnology. 13(6): 75-84.
- El sharabasy S.F., Bosila, H.A., Abdel-Aal, W.B., Mansour, B.M., Bana A.A. (2019). Effect of Vitamins (pyridoxine and nicotinic acid), Thiamine-Hcl and Myo-Inositol at Different Concentrations on Free Amino Acids and Indoles Content of Embryogenic Callus of in vitro Date Palm (Sakkoty and Bartamuda Cultivar) Materials Research Proceedings 11:244-252. Doi: https://doi.org/10.21741/9781644900178-20
- Gill, S.S. Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, 48(12): 909–930. Doi: https://doi.org/10.1016/j.plaphy.2010.08.016
- Habibi, N., Suthar, P. K. Purohit, S. D. (2009). Role of PGRs and inhibitors in induction and control of somatic embryogenesis in Themeda quadrivalvis. Indian Journal of Experimental Biology, 47(3): 198–203.
