Darush Choobineh ^1* Nafiseh Mahdinezhad ^1* Ali Niazi ² Baratali Fakheri ^1* Abbasali Emamjomeh ^1* Abdel Moneim E Sulieman ^3* Meshari Al-azmi ^4* Naimah Asid Alanazi ^3* Ahmed Issa Goniem ^3* Bahman Fazeli-Nasab ^5*

• ¹ Department of Plant Breeding and Biotechnology, College of Agriculture, University of Zabol, Zabol, IRAN, zabol, Iran
• ² Institute of Biotechnology, School of Agriculture, Shiraz University, Shiraz, Iran, shiraz, Iran
• ³ Department of Biology, College of Science, University of Haʼil, Haʼil, Saudi Arabia, Haʼil, Saudi Arabia
• ⁴ Bioinformatics Specialist, College of Computer Science and Engineering, University of Ha’il, Ha’il 81481, Saudi Arabia, Ha’il, Iran
• ⁵ Research Department of Agronomy and Plant Breeding, Agricultural Research Institute, University of Zabol, Zabol, Iran, Zabol, Iran

Introduction: Potato (Solanum tuberosum L.) is a crucial staple crop, ranking fourth in global food production after wheat, maize, and rice. Originating in the Andes, it is now grown in over 100 countries and is valued for its nutritional benefits and industrial uses, including starch production and processed foods like French fries. To address challenges from biotic and abiotic stresses, innovative genetic manipulation techniques like Agrobacterium-mediated transformation are vital for developing resilient potato cultivars.Objectives: HVA1 encodes a protein from the LEA III superfamily, essential for abiotic stress responses. This study presents a protocol for Agrobacterium-mediated transient transformation in planta, involving direct injection of a bacterial suspension into potato tuber sprouts. This method transforms HVA1 and EPSPS genes, conferring resistance to cold and glyphosate herbicide. Methods: The study utilized Agria potatoes from SPII, Iran, which were disinfected and germinated in a controlled environment. Agrobacterium tumefaciens strain C58 facilitated the transformation of potato sprouts, injected with a suspension containing Acetosyringone. The hva1 gene from Hordeum vulgare was cloned into a modified pBI121 vector for transformation. Transgenic plants were tested for glyphosate tolerance across various concentrations, with morphological changes monitored and analyzed statistically. Molecular analysis confirmed the presence and expression of the transgene through PCR and RT-PCR. Protein extraction from positive transformants was followed by ELISA testing to quantify transgene expression levels.In this study, we successfully generated and cloned a construct containing the hva1 gene into the pBI121 vector, which was then transformed into E. coli and Agrobacterium. Fifty treated tubers were transformed using the hva1-pBI121 construct. Subsequent experiments showed that transgenic potato plants exhibited Glyphosate tolerance, with 46% survival at 2% Glyphosate concentration, while nontransformed controls perished. PCR analysis confirmed stable integration of the transgenes hva1 and EPSPS in 18 out of 23 selected plants, with detectable expression levels verified through RT-PCR. ELISA assays indicated significant expression of the hva1 protein in transgenic plants compared to controls, with no adverse effects on growth or tuberization observed. Overall, the results demonstrate successful transformation and expression of the hva1 gene in potato plants.