Construction of genetic transformation system mediated by Agrobacterium rhizogenes in Vaccinium corymbosum

【Objective】Molecular breeding technology enables targeted improvement and innovation in blueberry germplasm, accelerating the development of superior blueberry varieties. The current genetic transformation system for blueberries faces limitations, including complex steps, time-consuming processes, and reliance on tissue culture systems. The Regenerative Activity-dependent in Planta Injection Delivery (RAPID) method enables the generation of stable transgenic plants without the need for tissue culture, offering broader applicability. Inspired by the RAPID method, we established an Agrobacterium rhizogenes transformation system for blueberry (Vaccinium corymbosum) stem segments.【Methods】 The stem segments from current-year green shoots of field-grown Lanmei 1 blueberry plants were used as explants. A. rhizogenes strain K599, carrying a green fluorescent protein (GFP) overexpression plasmid, was employed for transformation. The bacterial suspension was aspirated and injected into the bud points and both cut ends of the explants using a 1 mL sterile syringe. At the same time, the screening experiments were conducted to optimize the concentration of the bacterial suspension and the combination of two additives (acetosyringone, AS; surfactant L-77, S L-77). The infected explants were transplanted into substrate soil and subjected to dark cultivation for 4 days. After 45 days, GFP fluorescence signals in the infected plantlets were observed under 488 nm blue light using a stereo fluorescence mi-croscope. The potential positive transgenic plants were screened based on GFP expression. Subsequently, DNA was extracted from newly emerged lateral buds exhibiting green fluorescence signals, and GFP gene fragments were cloned by polymerase chain reaction (PCR) to identify the integration of the target gene into the blueberry genome.【Results】The survival rate of explants increased by approximately 10% when the concentration of A. rhizogenes was OD600 = 1.0 and 1.5, compared with that of OD600 = 0.5. Concurrently, when OD600 = 1.0, the induction rate of GFP- positive shoots increased by approximately 8% compared with the induction rates observed at OD600 = 0.5 and 1.5. Therefore, the optimal bacterial suspension concentration for the genetic transformation system was determined to be OD600 = 1.0. The different additive combinations (①100 µmol·L-1 AS, no S L-77; ②200 µmol·L-1 AS, no S L- 77; ③100 µmol·L-1 AS, 0.05% S L-77; ④200 µmol·L-1 AS, 0.05% S L-77) were added to the bacterial suspension, and the explants were infected by injection. The results showed that the survival rate of the explants was the highest in combination ①, reaching 87.5%, while the GFP- positive shoot induction rate was only 10.00%; and the survival rate of explants was the lowest in combination ②, at 80.00%, while the GFP-positive shoot induction rate was 30.00%, indicating that within the experimental dosage range, increasing the concentration of AS would improve the transformation efficiency but reduced the explant survival rates. In addition, the survival rate of the explants was 82.50% in combination ③, while the GFP- positive shoot induction rate was 40.00% ; and the survival rate of the explants was 80.00% in combination ④, while the GFP-positive shoot induction rate was 42.50%, indicating that the addition of surfactants would significantly enhance the transformation efficiency, outperforming the effects of increasing AS concentration alone. It is noteworthy that the improvement in transformation efficiency by adding 0.05% S L-77 diminished, as the AS concentration increased. The additive combination for the genetic transformation system was determined to be 200 µmol·L-1 AS with 0.05% S L-77. It was observed by cutting and cultivating the infected explants that the lateral buds began to sprout from the top of the stem segment 15 days later. By 45 days, nearly all surviving cuttings had sprouted, with no adventitious roots emerging around the bud sites. The callus formation was observed at the cut ends of some cuttings. The hairy roots mainly emerged from the basal ends of the stem segments, with rooting occurring at a slower rate compared with the lateral bud sprouting. It was found by observing the cuttings under an excitation light source of 488 nm that GFP fluorescence signals were expressed on both the newly formed lateral buds and hairy roots. And the GFP signal emitted by the leaves was relatively weak. The DNA of potential positive lateral buds was extracted and the transformation was verified by PCR. The results showed that the false positive rate of fluorescence screening was 30%.【Conclusion】A simple, rapid, and economical genetic transformation system for stem segments of Lanmei 1 blueberry, mediated by A. rhizogenes K599, was established in this study. The optimal conditions for the genetic transformation system are as follows: an Agrobacterium suspension with OD600 = 1.0 is used, supplemented with a combination of 200 µmol · L- 1 AS and 0.05% S L- 77 as additives, and the bud points and both ends of the green stem segments are infected via injection, followed by dark cultivation for 4 days. The fluorescence screening could be conducted approximately 45 days after cultivation. The achieved highest transformation efficiency was 29.75%. This genetic transformation system for blueberries seems to be capable of transforming blueberry varieties with no establishment of a tissue culture system, and should provide technical support for functional gene research and elite cultivar breeding in blueberries.