Establishment of an in vivo transgenic hairy root system in strawberry for verifying the nitrate-transport activity of FaNRT1.1

The study of strawberry gene function has been hindered by the low transformation efficiency and long generation time of transgenic plants. This study aimed to develop and optimize methods for generating strawberry (Fragaria × ananassa Duch. ‘Benihoppe’) plants with transgenic hairy roots. This involved inducing hairy roots on strawberry stolons near new plants, optimizing several parameters that affect the survival rate of stolon hairy roots, and using the new hairy root transgenic system to investigate the nitrate-transport function of FaNRT1.1. In vivo injection (IVI) of Agrobacterium rhizogenes K599 (OD₆₀₀ = 1.0) at sites measuring 0.5–1 cm on the tops of mature stolons (9–12 days old) of ‘Benihoppe’ strawberry resulted in the establishment of strawberries with transgenic hairy roots. The IVI induced epidermal bulges and hairy roots in 50% of the stolons. The epidermal bulges were evident, and callus began to grow 35 days post-injection, while hairy roots began to develop near the injection sites at 40 days and became abundant by 60 days. Specific fluorescence signals were observed in all transgenic hairy roots of 40 new plants. Using ¹⁵N nitrate labeling, we confirmed the nitrate-transport—from roots to shoots—function of FaNRT1.1 in the strawberry plants with transgenic hairy roots. Taken together, sufficient hairy roots can be induced using an efficient transgenic hairy root system, which can be effectively applied to gene function research, such as the analysis of the nitrate-transport activity of FaNRT1.1.