Optimization of In Vitro Regeneration in Wheat and Agrobacterium-mediated Rapid Transformation

Wheat ( Triticum aestivum L.) is a staple food that plays a significant role in human nutrition due to its high nutritional value. The development of an effective in vitro regeneration system is a prerequisite for achieving rapid Agrobacterium-mediated genetic transformation in wheat. In this study, the tested concentrations and combinations of picloram, 2,4-D, dicamba, and IAA in mature embryo cultures of Kırik and Palandöken 97. While analyzing CFR (%) and CW (%) and gene expression for 6 different media, ECR (%), RECR (%), RE (number), and the effects of 12 hormone-free and IAA-supplemented media were evaluated following somaclonal variation. Furthermore, following gene transfer optimization, the pGFPGUS Plus vector was introduced into 10-day-old calli via “ Agrobacterium -mediated transformation,” and HPTII , GUS , and mRNA expression were analyzed by qRT-PCR in selected T ₀ plants. According to the results, MS4-1 and MS1-1 generally had the greatest effects on ECR, RECR, and RE among the media, while MS2-1 and MS2-2 generally had the least. In both varieties, the WOX4 gene response peaked at 14 DAI and decreased by 28 and 42 DAI. LEC1 expression peaked at 42 DAI, while the BBM1 and TaSERK genes showed high expression at 14 and 28 DAI, respectively. MS4 was selected as the most effective medium for all genes. In both Kırik and Palandöken-97, the MS1-1, MS3-1, and MS4-1 media showed the closest similarity to the parent plant. As a result of gene transfer, 12 To plants were selected. Line 11 exhibited the highest response in both GUS and HPTII. Expression levels were higher than in the control in all 12 lines. Overall, the tests revealed that Kırik and MS4 emerged as effective media for rapid gene transfer. Optimized in vitro regeneration and transformation protocols enhance the effectiveness of biotechnological approaches in wheat breeding, thereby making significant contributions to sustainable agriculture.