PROTOCOL OPTIMIZATION FOR EMBRYO RESCUE: IMMATURE EMBRYO CULTURE OF WHEAT AND VARIETAL PERFORMANCE ANALYSIS FOR CALLUS INDUCTION

Abstract

Wheat (Triticum aestivum L.) is one of the most important staple crops worldwide, which is now under threat of productivity loss caused by changing climates, genetic erosion, and increasing biotic and abiotic stresses. The development of genetic and biotechnological solutions depends greatly on the establishment of highly efficient cell culture and plant regeneration systems. In this study, the protocol for tissue culture propagation of embryo rescue was optimized, and the callus induction efficiency of 4 different wheat genotypes, Sehar-2006, Galaxy-13, Sawera-2024, and Falak-2025, was assessed. Immature embryos were dissected and surface sterilized with a sequential treatment of sodium hypochlorite, Tween-20, and 70% ethanol. Murashige and Skoog (MS) basal medium containing 30 g L⁻¹ sucrose, 2 g L⁻¹ Phytagel, and 3 mL L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) stock solution was used for the culture of the sterilized explants. After a 2-week incubation period under controlled environmental conditions, there were considerable genotypic differences in terms of callus induction performance. Falak-2025 showed the highest callus induction and embryo rescue success rate (~78%), which was followed by Sawera-2024 with callus induction of ~70% and embryo rescue of ~50%, whereas Galaxy-13 showed callus induction of ~45% and embryo rescue of ~45%. The results obtained validate that the immature embryos are a good explant source because of their high metabolic activity and high regeneration potential. In addition, the application of the exogenous auxin 2,4-D was essential for the initiation of embryogenic callus formation. Finally, the optimization of all the above-mentioned aspects (genotypic selection, stage of explant culture, and hormonal balance) plays a crucial role in the success of wheat embryo culture. The improved protocol described in this study offers a reliable and repeatable callus induction system that could be used in subsequent wheat improvement projects, such as genetic transformation and producing stress-tolerant cultivars.