Optimization of In-vitro Androgenesis in Cereals: Role of Temperature Conditions and Iron Availability

Abstract

Background and Aims: Sorghum (Sorghum bicolor L.) is a climate-resilient cereal crop widely valued for its high productivity and adaptability to drought, heat, and low-fertility soils, making it crucial for sustainable agriculture and food security. The aim of this study was to optimize in vitro anther culture conditions in sorghum (Sorghum bicolor L.) by evaluating the effects of different iron sources and temperature regimes on callus induction, embryogenic response, and plant regeneration efficiency. The influence of two iron formulations, Fe-EDTA and ferric citrate, on anther growth, viability, callus induction, and morphogenetic response was assessed using Gamborg B5 agar medium. In addition, the effects of optimal, elevated, and reduced temperature conditions on sorghum growth dynamics were analyzed. In the context of global climate change and the increasing demand for stress-tolerant cereal genotypes, the development of effective protocols for haploid and doubled haploid production is of critical importance for modern breeding programs. Study Design:  Experimental in vitro study using a randomized design with iron source and temperature as treatment factors. Place and Duration of Study: The study was conducted at the Plant Biotechnology Laboratory, Satbayev University, from May 2025 to March 2026. Methodology: Sorghum anthers were cultured under controlled in vitro conditions on induction media supplemented with Fe-EDTA or Fe-citrate as iron sources. Cultures were maintained under standardized photoperiod and temperature regimes, including optimal (25–34°C), heat stress (>34°C), and cold stress (≤20°C) conditions. Callus induction frequency, embryogenic response, plant regeneration efficiency, and relative growth indices were recorded and statistically analyzed. Results: The type of iron source significantly influenced in vitro response. Fe-EDTA supplementation resulted in the highest callus induction frequency (78.4 ± 2.3%), compared to Fe-citrate (61.2 ± 1.8%). Plant regeneration efficiency was also greater under Fe-EDTA treatment (42.7 ± 1.5%) than under Fe-citrate (28.9 ± 1.3%). Under optimal temperature conditions (25–34°C), sorghum exhibited the highest relative growth index (100 ± 3.1%), while heat stress (>34°C) reduced growth to 72.5 ± 2.7%, and cold stress (≤20°C) to 54.8 ± 2.1%, confirming the strong inhibitory effect of temperature stress on sorghum growth. Conclusion: The findings demonstrate that both iron formulation and temperature regime are critical determinants of successful in vitro androgenesis in sorghum. Fe-EDTA was identified as the most effective iron source for enhancing callus induction and plant regeneration, while optimal temperature conditions maximized growth performance. These results contribute to the refinement of cereal androgenesis protocols and may improve doubled haploid production efficiency for climate-resilient breeding strategies.