Establishment of genetic transformation system mediated by Agrobacterium rhizogenes in strawberry

【Objective】The study aimed to establish an efficient, economical and rapid Agrobacterium rhizogenes-mediated transformation system in strawberry.【Methods】This study used four strains of A. rhizogenes, namely Ar.1193, Ar.Qual, C58C1, and K599, and selected octoploid cultivated strawberry Fenyu and diploid woodland strawberry rg40 and Ruegen as experimental materials. The study adopted scratching stolon method, puncturing shortened stem method and hypocotyl infection method and 35S:: RUBY visual reporter gene to systematically evaluate the hairy root transformation efficiency of different strains and to screen the strain for optimal transformation. For scratching stolon method, Fenyu stolon-derived plantlets were used as the experimental material. The A. rhizogenes carrying the 35S::RUBY plasmid was streaked onto YEP solid medium and cultured at 28 ℃ for 2-3 days. The primary roots of the stolon-derived plantlets were removed, and the base of the stolon was lightly wounded with a sterile blade. The solid bacterial cells were then applied to the wounded area. The sterilized vermiculite was placed into transparent plastic cups. The treated stolon- derived plantlets were inserted into the cups, which were subsequently wrapped with aluminum foil to simulate a dark environment for root growth. The remaining bacterial cells on YEP solid medium were resuspended in water to create a bacterial suspension, which was used to irrigate the wounded area. The infected stolon-derived plantlets were cov-ered with a transparent cover to maintain high humidity and were wrapped with black cloth for overnight dark treatment. The next day, the black cloth was removed, and the infected stolon-derived plantlets were kept in a high-humidity environment. After 20 days of insertion, the stolon-derived plantlets were uniformly observed. For puncturing shortened stem method, one-month-old woodland strawberry rg40 were used as the experimental material. The A. rhizogenes carrying the 35S::RUBY plasmid was streaked onto YEP solid medium and cultured at 28 ℃ for 2-3 days. A single colony from the plate was picked using a syringe needle and used to perform multiple piercings at the short stem region of the seedling. Subsequently, the bacterial cells were scraped from the YEP solid medium and applied to the short stem region. The seedlings were then planted in the substrate. Since the diploid seedlings were relatively tender, it was not convenient to pull them up for observation at an early stage. Therefore, observation was conducted after 45 days. For hypocotyl infection method, the A. rhizogenes carrying the 35S::RUBY plasmid was streaked and cultured on YEP solid medium for 2-3 days to obtain single colonies. A positive single colony was selected and inoculated into YEP liquid medium containing 75 mg·L-1 spectinomycin and 50 mg · L- 1 streptomycin, and cultured at 28 ℃ with shaking at 220 r · min- 1 for 16 hours. The next day, the culture was inoculated at a 1∶100 ratio into 50 mL of YEP liquid medium containing 75 mg · L- 1 spectinomycin and 50 mg · L- 1 streptomycin, and cultured at 28 ℃ until the OD600 reached 0.6. The bacterial suspension was collected by centrifugation at 3220 g for 10 minutes and resuspended in 1/2MS liquid medium containing 100 µmol·L- 1 acetosyringone (AS) to achieve an OD600 of 0.8. The bacterial suspension was activated at 28 ℃ with shaking at 100 r·min-1 for 1 hour. When the Ruegen seedlings had two cotyledons, the hypocotyl was cut off from the root, and the hypocotyl with cotyledons was used as experimental material. The explant was immersed in the Agrobacterium suspension and cultured at 28 ℃ with shaking at 100 r·min-1 for 15 minutes, followed by sonication for 5 seconds (KQ-500DE digital ultrasonic cleaner, power 90%). The infected explant was then transferred to a filter paper moistened with 1/2MS (containing 2% sucrose and 100 µmol·L-1 AS) and co-cultured in the dark at 23 ℃ for 4 days. After co-cultivation, the infected explant was washed with sterile water and transferred to 1/2MS medium containing 2% sucrose 250 mg·mL-1 cefotaxime sodium (CEF) for decontamination and hairy root induction. The explants were observed uniformly 30 days after infection.【Results】The results showed that under the three different treatment methods, all four strains of A. rhizogenes could induce the strawberry explants to produce hairy roots. Among the four strains, the strain Ar.1193 showed the best positive rooting rate. In the scratching stolon method, the induction rate of positive hairy roots for the octoploid cultivated strawberry Fenyu reached 79.41%. The induction rate of positive hairy roots for the diploid woodland strawberry rg40 was 73.33% using the puncturing shortened stem method, while the induction rate of positive hairy roots for the diploid woodland strawberry Ruegen was 14.38% via the hypocotyl infection method.【Conclusion】Through three methods, Ar.1193 was identified as the most suitable strain for inducing strawberry hairy roots. This study preliminarily established a strawberry hairy root culture system using the 35S::RUBY reporter gene for screening. This system not only would provide a scientific basis for the subsequent genetic transformation of strawberries but also would lay the foundation for the rapid validation of strawberry root development and CRISPR/Cas9 gene editing efficiency.