Sodium Bicarbonate and Hydrogen Peroxide Irrigation Improves (Ex Vitro) Survival of Agrobacterium-Transformed Wheat. Plantlets: A Comparative Assessment of Post-Regeneration Interventions: Na₂CO₃ Plus H₂O₂ Irrigation for Survival of Agrobacterium-Transformed Wheat

Muhammad Tahir Arshad

doi:10.54393/fbt.v6i2.244

Authors

Muhammad Tahir Arshad Department of Biotechnology, Forman Christian College, University, Lahore, Pakistan

DOI:

https://doi.org/10.54393/fbt.v6i2.244

Keywords:

Triticum aestivum, Agrobacterium-Mediated Transformation, Ex Vitro Acclimatization, Sodium Bicarbonate, Hydrogen Peroxide

Abstract

Low ex vitro survival rates of transgenic wheat plantlets following transfer from tissue culture to soil represent a major constraint limiting the efficiency of genetic transformation pipelines, with losses often exceeding 70-90%. Objectives: To compare the effectiveness of four simple, low-cost post-regeneration interventions PPM supplementation, photo-acclimatization, modified root-transfer technique, and irrigation with sodium bicarbonate Na₂CO₃ + H₂O₂ in improving the ex-vitro survival of Agrobacterium-transformed Borlaug-16 wheat plantlets, and to identify the most practical protocol modification for routine transformation workflows. Methods: Fifty putative transgenic wheat plantlets (Triticum aestivum L. cv. Borlaug-16), generated via Agrobacterium tumefaciens (strain AGL1)-mediated transformation of immature embryo-derived calli with a Bar gene selection cassette, were allocated to four treatment groups (n=10 each) and one untreated control (n=10). Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method, and PCR analysis targeting the ubiquitin promoter–Bar gene junction confirmed transgene integration in all surviving plantlets. Results: The Na₂CO₃ + H₂O₂ irrigation treatment achieved the highest survival rate at 30%, representing a 20-percentage-point improvement over the untreated control (10%). In contrast, PPM supplementation showed no improvement (10%), while both photo-acclimatization and modified root-transfer resulted in 0% survival. PCR analysis confirmed the presence of the expected ~1 kb ubiquitin–Bar amplicon in all surviving plantlets. Conclusions: Supplementing irrigation water with Na₂CO₃ and H₂O₂ provides an inexpensive and effective strategy for ex-vitro survival of transgenic wheat plantlets, whereas PPM, photo-acclimatization, and tissue-wrapped root transfer offered no advantage under the conditions tested.

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