Establishment of Agrobacterium-Mediated Transient Transformation System in Sunflower

Sunflower (<i>Helianthus annuus</i> L.) is an important oilseed crop in Northwest China, exhibiting resistance to salt and drought. Mining its excellent tolerance genes can be used for breeding. However, the current platforms for identifying gene function in sunflower is inadequate. The transient transformation system, which can rapidly validate gene function, shows promising prospects in research. In this study, we established an efficient transient expression transformation system for sunflower using three methods: <i>Agrobacterium</i>-mediated infiltration, injection, and ultrasonic-vacuum. The detailed procedures were as follows: <i>Agrobacterium</i> GV3101 carrying a <i>GUS</i> reporter gene on the pBI121 vector with an OD₆₀₀ of 0.8 as the bacterial suspension and 0.02% Silwet L-77 as the surfactant were utilized in all three approaches. For the infiltration method, seedlings grown hydroponically for 3 days were immersed in a bacterial suspension containing 0.02% Silwet L-77 for 2 h; for the injection method, the same solution was injected into the cotyledons of seedlings grown in soil for 4 to 6 days. Subsequently, the seedlings were cultured in the dark at room temperature for three days; for the ultrasonic-vacuum method, seedlings cultured in Petri dishes for 3 days were first subjected to ultrasonication at 40 kHz for 1 min, followed by vacuum infiltration at 0.05 kPa for 5-10 min. <i>Agrobacterium</i>-mediated transient transformation efficiency achieved by the three methods exceeded 90%, with gene expression being sustained for at least 6 days. Next, we employed the infiltration-based sunflower transient transformation technology with the <i>Arabidopsis</i> stable transformation platform to confirm salt and drought stress tolerance of candidate gene <i>HaNAC76</i> from sunflower responding to various abiotic stresses. Altogether, this study successfully established an <i>Agrobacterium</i>-mediated transient transformation system for sunflower using these three methods, which can rapidly identify gene function and explore the molecular mechanisms underlying sunflower's resistance traits.