Agrobacterium Rhizogenes-Mediated Transformation of Antifreeze Protein in Tobacco Hairy Roots

Saleci, Milad; Khorshidi, Mehdi; Davarpanah, Seyed Javad

doi:10.30491/jabr.2026.557223.1939

Agrobacterium Rhizogenes-Mediated Transformation of Antifreeze Protein in Tobacco Hairy Roots

Document Type : Original Article

Authors

Milad Saleci ¹

Mehdi Khorshidi ²

Seyed Javad Davarpanah ¹

¹ Applied Biotechnology Research Center, New Health Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

² Department of Plant Sciences, Faculty of Biology, Damghan University, Damghan, Iran

10.30491/jabr.2026.557223.1939

Abstract

Introduction: Antifreeze proteins (AFPs) decrease the freezing point, not the melting point, of aqueous solutions through inhibition of ice crystal growth by an adsorption-inhibition mechanism. AFPs have many applications in cryopreservation. Nevertheless, low yield and high extraction cost of extracting AFPs from natural sources limit their wide applications. To overcome this limitation, here we genetically transformed tobacco hairy roots mediated by Agrobacterium rhizogenes.
Materials and Methods: Recombinant vector was transformed into Agrobacterium rhizogenes strain 15834 by heat-shock method. Nicotiana tabacum leaf discs were co-cultured with recombinant Agrobacterium rhizogenes under vacuum condition. PCR was used to examine whether transgenic lines were free from A. rhizogenes and to confirm integration of AFP gene. Western blotting was performed to confirm AFP expression.
Results: Using this method, we achieved a high transformation efficiency of 86.7%. Molecular analysis confirmed the integration of the AFP gene and the absence of A. rhizogenes contamination in the hairy root lines. Western blotting confirmed a high level of AFP expression, with a molecular mass of approximately 20 kDa. After 30 days of cultivation in flasks, a hairy root biomass of approximately 4.9 g was obtained.
Conclusions: Tobacco hairy root expression platform offers a fast, low-cost, and efficient system for heterologous production of antifreeze proteins and should be considered to employ in industrial scales in the future.

Keywords

Antifreeze Protein

Heterologous Expression

Agrobacterium rhizogenes

Hairy Root

Nicotiana tabacum