Co-Transformation of Persian Walnut Somatic Embryos with Fld and BADH Genes for Enhanced Drought and Salinity Tolerance

Salinity and drought are among the most significant challenges in agriculture, adversely affecting plant growth, including that of Persian walnut ( Juglans regia ). Genetic engineering offers a promising approach to improving Persian walnut resistance to these environmental stresses by introducing stress-tolerance genes such as betaine aldehyde dehydrogenase ( BADH ) and flavodoxin ( Fld ). In this study, somatic embryos of J. regia cv. Chandler were inoculated with Agrobacterium tumefaciens strain LBA4404, which harbors the binary plasmid pBI121. This plasmid contains the Fld and BADH genes, regulated by the ubiquitin (444I) and CaMV35S promoters, respectively, along with the NPTII gene as a selectable marker under the control of the NOS promoter. Following inoculation, putative transgenic embryos were selected on antibiotic-containing medium and directed towards germination and plantlet regeneration. Polymerase chain reaction (PCR) analysis confirmed the successful integration of the transgenes in lines 4, 6, 10, and 12, achieving a transformation efficiency of 4.44%. Furthermore, reverse transcription PCR (RT-PCR) demonstrated the successful expression of both target genes in these four lines. The transgenic plantlets (lines 4, 6, 10, and 12), along with wild-type controls, were subjected to salt (0, 50, 100, and 200 mM NaCl) and osmotic stress (0%, 5%, 10%, and 15% polyethylene glycol (PEG)) treatments for 30 days. The results indicated that transgenic plantlets exhibited significantly higher tolerance compared to wild-type controls, as evidenced by increased survival rates, larger callus diameters, higher healthy leaf counts, greater plant heights, and elevated proline and glycine betaine (GB) content. Among the transgenic lines, line 4 demonstrated the highest level of stress tolerance. Additionally, transgenic lines exhibited the highest levels of proline and GB under osmotic and salinity stress conditions. Overall, the simultaneous introduction of the Fld and BADH genes enhanced photosynthetic efficiency (due to Fld ) and improved osmotic regulation (due to BADH ), thereby conferring resistance to drought and salinity stresses. These findings suggest that co-transformation with these genes presents a promising strategy for enhancing environmental stress tolerance in Persian walnut.