Agrobacterium-mediated genetic Transformation system of somatic embryo in Walnut (Juglans regia L.)

Abstract Walnut ( Juglans regia L.), also known as English or Persian Walnut, is a critical germplasm resource with significant ecological and economic value. Traditional walnut breeding strategies usually require a long time, and the breeding efficiency is poor. However, biotechnology has the potential to overcome the limitations of conventional breeding and accelerate germplasm improvement. This study established and optimized efficient Agrobacterium -mediated genetic transformation conditions for walnut. Somatic embryo (SE) of the walnut cultivar 'ZL-6' was induced as the transgenic recipient material. The Agrobacterium tumefaciens strain GV3101 was utilized to introduce plant expression vectors containing YFP and GUS reporters for the somatic embryogenesis transformation. This study systematically evaluated the impact of different factors on the transformation efficiency of somatic embryogenesis (SE), including the duration of preculture for SE, Agrobacterium cell density, and inoculation duration in the transformation system. The orthogonal test showed that the preculture for SE was a critical limiting factor for efficient Agrobacterium -mediated delivery. At the same time, the effects of Agrobacterium cell density and inoculation duration on transformation efficiency were not significant. The optimal Agrobacterium -mediated genetic transformation conditions for ‘ZL-6’walnut SE was: preculture of SE for about one month to reach the competent state, and Agrobacterium cell density with an OD 600 = 0.6 inoculating for 15 min. This Agrobacterium -mediated transformation system for walnut provides a platform for genetic improvement of core germplasm and further verification of gene function.